Anti-CD26 antibodies and uses thereof

ABSTRACT

The present invention pertains to novel antibodies capable of binding to CD26, as well as to their use as a medicament. Moreover, the present invention provides antibodies for use in treating and/or preventing Graft-versus-Host Disease (GvHD), for use in treating Aplastic Anemia and/or for use in promoting engraftment after haematopoietic stem cell transplantation. Furthermore, the present invention provides pharmaceutical compositions comprising at least one antibody of the present invention, as well as provides a kit of parts.

FIELD OF THE INVENTION

The present invention pertains to novel antibodies capable of binding toCD26, as well as to their use as a medicament. Furthermore, the presentinvention relates to antibodies for use in treating and/or preventing atleast one of Graft-versus-Host Disease (GvHD) and Aplastic Anemia, aswell as to antibodies for use in promoting engraftment afterhaematopoietic stem cell transplantation.

BACKGROUND OF THE INVENTION

CD26 is a widely distributed 110 kDa cell surface glycoprotein,initially defined as a T-cell activation antigen (Fox et al. (1984) J.Immunol. 133, 1250-1256, Fleischer (1987) J. Immunol. 138, 1346-1350,and Morimoto et al. (1989) J. Immunol. 143, 3430-3439). This moleculehas been shown to have dipeptidyl peptidase IV (DPPIV; EC3.4.14.5)activity in its extracellular domain, and wide tissue distribution(Hegen et al. (1990) J. Immunol. 144, 2908-2914 and Ulmer et al. (1990)J. Immunol. 31, 429-435; WO 2007/014169 A2) CD26 has multiple functionsin human T-cell physiology. For instance, evidence suggests that CD26can deliver a costimulatory signal for T-cell activation (Morimoto etal. (1994) Immunologist 2: 4-7 and Fleischer (1994) Immunol. Today15:180-184). Further, CD26 has been identified as the ADA bindingprotein, and the CD26/ADA complex may play a key role in regulatingimmune system function (Dong et al. (1996) J Immunol. 156(4):1349-55,Kameoka et al. (1993) Science. 261(5120):466-9, and Morrison et al.(1993) J Exp Med. 177(4):1135-43). A functional association between CD26and the cellular protein topoisomerase II α has also been reported(Aytac et al. (2003) British Journal of Cancer 88:455-462). Anti-CD26antibodies are e.g. known from WO 2007/014169 A2.

Haematopoietic stem cell transplantation (HSCT) represents an importanttherapy for many haematological and numerous epithelial malignancies, aswell as for a considerable number of non-malignant diseases (Ferrara etal., 2009, Lancet.; 373: 1550-1561; Sun et al., 2007, Transl. Res.; 150:197-214). Graft-versus-host disease (GvHD) is a major complication ofallogeneic haematopoietic stem cell transplantation (HSCT), andtherefore limits the use of these important therapies.

There are two major types of haematopoietic cell transplantation:autologous and allogeneic. Autologous transplantation involves isolationof haematopoietic stem cells (HSC) from a patient, storage of the stemcells, medical treatment of the patient that destroys stem cellsremaining in the body, and return of the patient's own stored stem cellsto his body. Autologous transplants have the advantage of a lower riskof graft rejection, infection and other correlated diseases. Allogeneictransplantation involves two persons: one is the healthy donor and oneis the patient or recipient. Allogeneic HSC donors must have a tissue(HLA human leukocyte antigens) type that matches the recipient and, inaddition, the recipient requires immunosuppressive medications. Thereare three possible sources of haematopoietic stem cells fortransplantation: the Bone Marrow (BM), the Peripheral Blood (PB) and theUmbilical Cord Blood (UCB).

The development of novel strategies has helped to expand the indicationsfor allogeneic HSCT over the last several years (Sun et al., 2007,supra). Improvements in infectious prophylaxis, immunosuppressivemedications, supportive care and DNA-based tissue typing have alsocontributed to improved outcomes after allogeneic HSCT (Ferrara et al.,2009, supra). For these reasons, the number of allogeneic haematopoieticcell transplantations continues to increase. However, graft-versus-hostdisease (GvHD) remains a major complication of allogeneic HSCT.

GvHD occurs when donor T cells identify genetically defined proteins onhost cells as not-self and mount an immune response in order to destroythem (Ferrara et al., 2009, supra). Depending on the time at which itoccurs after HSCT, GvHD can be either acute or chronic. Acute GvHD(aGvHD) is responsible for 15% to 40% of mortality and is the majorcause of morbidity after allogeneic HCT, while chronic GvHD (cGvHD)occurs up to 50% of patients who survive three months after HCT (Sun etal., 2007, Transl. Res.; 150: 197-214).

Acute Graft-versus-Host Disease generally occurs after allogeneic HSCTas reaction of donor immune cells against host tissues. The three maintissues affected by acute GvHD are the skin, liver, and gastrointestinaltract. Clinically, the diagnosis is suspected when a recipient of HSCTdevelops any or all of the following signs or symptoms: dermatitis (skinrash), cutaneous blisters, crampy abdominal pain with or withoutdiarrhoea, persistent nausea and vomiting, hepatitis (with elevation ofbilirubin and/or liver enzymes). Symptoms most frequently start withdonor engraftment, before day 100 after the HSCT, but may also occurlate. Acute GvHD is a clinical diagnosis confirmed by histologicalevidences.

Acute GvHD can be staged by the number and extent of organ involvement.The current staging system is derived from Glucksberg first aGvHDclassification in 1974 (Glucksberg et al., 1974, Transplantation; 18:4295-304). Recent data support the use of the grading system, since it isable to subdivide patients into risk categories for complications andmortality. In this system, patients are divided into one of four grades(I-IV) depending on the degree or stage of involvement in three organs.The skin is staged with percent body surface involved, the liver isstaged with degree of bilirubin elevation, and the gastrointestinaltract is staged with amount of diarrhoea. Using these criteria, a singlegrade is assigned to each patient (Jacobsohn et al., 2007, Orphanet J.of Rare Diseases; 2:35).

Various clinical manifestations of GvHD are known. The earliest and mostcommon manifestation is skin GvHD. This is essentially a maculopapularrash that can begin anywhere in the body but often start with palm andsole involvement. The patient may complain of pruritus or tenderness inaffected areas. In severe cases, blisters may occur. Thegastrointestinal manifestations include diarrhoea, which may becomebloody, cramping, nausea, vomiting and failure to thrive. Furthermore,jaundice from hyperbilirubinemia is the hallmark of liver GvHD(Jacobsohn et al., 2007, supra), although a hepatitic variant of GvHDwith a rise in liver enzymes like an acute viral hepatitis, has beenrecognized (Akpek et al., 2002, Blood; 100: 3903-3907). Even ifmethylprednisolone is not registered in any European Countries for thisindication, it is considered current standard of care in first linetreatment of acute GvHD.

First line treatment of acute GvHD, with methylprednisolone 2 mg/kg/dayis effective in over 50% of patients, but produces durable responsesonly in ⅓ of the patients. Non responders are offered second linetherapy, which is based on combinations of immunosuppressive agents notregistered in this indication. Second line therapy is largelyunsatisfactory with one year survival of 30% in most large clinicaltrials. None of these strategies has achieved the level of successrequired to become standard of care. After 30 years of transplantexperience steroid refractory acute GvHD (aGvHD), remains largely anuntreatable disease. It has to be emphasized that aGvHD patientsresistant to steroid therapy have very limited therapeutic options andthat there are no currently authorized treatments for this clinicalsituation. This condition is life-threatening in particular due to theincreased mortality in this patient population, particularly secondaryto infection.

Any clinically relevant result in this patient population would be ofsignificant benefit as it would offer a clinically relevant advantagefor steroid resistant aGvHD patients.

Moreover, approaches for facilitating engraftment after haematopoieticstem cell transplantation, will be useful. Engraftment is the process inwhich the transplanted stem cells find their way to the bone marrowspaces in the centre of the large bones of the body. Only then can thetransplanted stem cells begin to produce new blood cells. Experts arenot completely certain how this process happens but it is generallyacknowledged that this is a long process: it takes approximately two tofour weeks after the bone marrow is infused for engraftment to occur.Until the blood stem cells engraft, the patient will be at risk ofdeveloping an infection. This is because the transplanted patient hasbeen normally subjected to radiation and/or chemotherapy, whose resultis the destruction of the white blood cells in the patient's body. Whilewaiting for the engraftment, a transplanted patient could suffer ofserious complication due to an infection (caused by bacteria, virus orfungus), which is one of the main cause of transplant related mortalityafter Bone Marrow Transplantation (BMT). Accordingly, there is a need inthe art for an agent able to improve engraftment. Such an agent will beof significant value for BM transplanted patients. If homing andengraftment can be enhanced, the time to recovery of hematopoieticlineages can be reduced resulting in less engraftment failures andbetter overall survival, especially in UCB transplantation (Broxmeyer.H. E. (2006). Umbilical Cord Blood Stem Cells: Collection, Processing,and Transplantation. Blood Banking and Transfusion Medicine: BasicPrinciples and Practice. C. D. Hillyer et al., Churchill Livingston, animprint of Elsevier. Inc.: 823-832: Lewis, 2002, Intern Med J 32(12):601-9).

Aplastic anemia is a type of anemia, wherein bone marrow fails toproduce sufficient amounts of blood cells for replenishing blood cells.In particular, a congenital and an acquired form of aplastic anemia mayexist. Acquired aplastic anemia (AA) is a rare bone marrow failure statecharacterized by marrow hypocellularity and low peripheral blood cellcounts [Young N S, Maciejewski J P. The pathophysiology of acquiredaplastic anemia. N Eng J Med 1997, 336:1365-1372]. The evidence of anautoimmune pathogenesis is mostly indirect and the characterization ofthe underlying immune response is incomplete mainly due to technicaldifficulties resulting from the disease-specific hypocellularity.Acquired Aplastic anemia is thought to be an immunomediated disease, andcurrent standard non transplant therapy is anti-thymocyte globulin (ATG)plus cyclosporin A (CsA). Failures include patients not responding tofirst line (30%) and patients relapsing after a first response (30%),such that event free survival does not exceed 30-40% (Bacigalupo A.,Passweg J., 2009, Hematol Oncol Clin North Am. 23: 159-70).

Untreated aplastic anemia may lead to death, in some cases even within ashort period of merely several months. Current treatments of aplasticanemia encompass for example bone marrow transplantation orimmunosuppressive drug therapies. Immunosuppressive drug therapies failhowever in a significant number of cases and bone marrow transplantationis not possible in absence of an appropriate donor. Thus, there is alsoa need in the art to provide alternative agent(s) for treating aplasticanemia, which preferably may be effective for treating patients whichare non-responsive to at least one other therapy.

BRIEF DESCRIPTION OF THE INVENTION

The present invention is concerned with the provision of an agent whichcan be used for treating and/or preventing disease(s), disorder(s) andcondition(s), in particular immune-system related disease(s),disorder(s) and condition(s). In particular, the present inventors aimedat the provision of an agent which may be used for treating and/orpreventing at least one of Graft-versus-Host disease (GvHD) and AplasticAnemia or which may be used for promoting engraftment afterhaematopoietic stem cell transplantation. Preferably, this agent shouldbe moreover essentially well tolerated by patients. In particular, thepresent inventors aimed at the provision of an agent which preventsand/or treats at least one of GvHD and Aplastic Anemia or promotesengraftment, in patients, in particular in one or more groups ofpatients, which are non-responsive to another treatment, in particularto another treatment with an immunosuppressive agent, for example atreatment with a steroid, or show an insufficient response thereto.

As a solution to these problems, the present inventors provide interalia an antibody, a pharmaceutical composition, an isolated nucleic acidmolecule, a vector, a composition comprising an antibody mixture, arecombinant host cell, a kit of parts and a process for manufacturing anantibody.

According to a first aspect, an antibody is provided, which antibody canspecifically bind CD26, in particular human CD26, said antibody cancomprise a heavy chain variable region and a light chain variableregion, wherein said heavy chain variable region can comprise thesequence WTWGPGYFDV (SEQ ID NO: 1), and/or wherein said light chainvariable region can comprise the sequence QQRSSYPNT (SEQ ID NO: 2)and/or the sequence GQGYSYPYT (SEQ ID NO: 3). Furthermore, an antibodyof the present invention can have a light chain variable region, whichlight chain variable region comprises an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 4, 5, 6 to 21, a variant of theamino acid sequence of SEQ ID NO: 4, and a variant of the amino acidsequence of SEQ ID NO: 5. Moreover, an antibody of the present inventionspecifically binds CD26 and can have a heavy chain variable region,which heavy chain variable region comprises an amino acid sequenceselected from the group consisting of SEQ ID NOs: 22 to 47 andvariant(s) thereof. In a particular embodiment, an antibody of thepresent invention specifically binds CD26 and can have a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 26 anda light chain variable region comprising the amino acid sequence of SEQID NO: 4 or SEQ ID NO: 5.

According to another aspect, the present inventors provide an isolatednucleic acid molecule comprising (a) a nucleotide sequence encoding anantibody of the present invention; or (b) a nucleotide sequencecomplementary to (a). According to still another aspect, an isolatednucleic acid molecule is provided, which comprises a nucleotide sequenceselected from the group consisting of SEQ ID NOs: 50 to 128 and avariant thereof, said variant having at least 90% sequence identity tothe nucleotide sequence selected from the group consisting of SEQ IDNOs: 50 to 128. According to yet another aspect, the present inventorsprovide an expression vector comprising a nucleic acid molecule of thepresent invention, wherein said nucleic acid molecule is operativelylinked to an expression control sequence. According to still anotheraspect, a recombinant host cell is provided, which comprises a nucleicacid molecule of the present invention.

According to yet another aspect, an antibody is provided, which isproduced from the hybridoma cell line deposited on the 11 Sep. 2012under the Budapest Treaty at the Centro di Biotecnologie Avanzate(CBA)—Interlab Cell Line Collection (ICLC) of Genoa (L. go R. Benzi, 10,Genoa, Italy) as PD 12002 or a derivative of said hybridoma cell line.The hybridoma cell line material deposited is also referred to hereinshortly as PD 12002 hybridoma deposit. All restrictions as to theavailability of these deposits will be withdrawn upon first publicationof this application or another application which claims benefit ofpriority to this application. According to still another aspect, thepresent inventors provide an antibody that binds the epitope bound by anantibody produced by the hybridoma cell line deposited at CBA-ICLC ofGenoa (Italy) as PD 12002.

According to yet another aspect, a process of manufacturing an antibodyof the present invention is provided.

According to still another aspect, the present inventors provide apharmaceutical composition comprising at least one antibody of thepresent invention and optionally at least one pharmaceuticallyacceptable excipient. According to yet another aspect, a pharmaceuticalcomposition of the present invention for use as a medicament isprovided. According to still another aspect, the present inventorsprovide a pharmaceutical composition of the present invention for use inpromoting engraftment after haematopoietic stem cell transplantation,and/or for use in preventing and/or treating Graft-versus-Host disease(GvHD), in particular after haematopoietic stem cell transplantation,and/or for use in preventing and/or treating Aplastic Anemia.

According to yet another aspect, an antibody or an antibody mixture ofthe present invention, in particular a composition comprising anantibody mixture of the present invention, for use as a medicament isprovided. According to still another aspect, the present inventorsprovide the antibody of the present invention or the antibody mixture,in particular the composition comprising an antibody mixture, for use inpromoting engraftment after haematopoietic stem cell transplantation,and/or for use in preventing and/or treating Graft-versus-Host disease(GvHD), preferably after haematopoietic stem cell transplantation,and/or for use in preventing and/or treating Aplastic Anemia, preferablySevere Aplastic Anemia. According to yet another aspect, a kit of partsis provided, which comprises: (i) at least one antibody of the presentinvention, in particular a composition comprising an antibody mixture ofthe present invention, and additionally (ii) a) at least oneimmunosuppressive drug or b) at least one corticosteroid and/or at leastone antihistamine.

DESCRIPTION OF THE FIGURES

FIG. 1a shows full CD26 human sequence.

FIG. 1b shows full CD26 porcine sequence.

FIG. 1c shows aligned full CD26 human and porcine sequence.

FIG. 2a shows CDR3 sequences, which can be present in an antibodyaccording to the present invention.

FIG. 2b shows the list of sequences of the VH CDR1, VH CDR2, VH CDR3, VLCDR1, VL CDR2, and VL CDR3 of CD26 specific antibodies.

FIG. 2c shows the list of sequences of the VH ABR1, VH ABR2, VH ABR3, VLABR1, VL ABR2, and VL ABR3 of CD26 specific antibodies.

FIGS. 3a and 3b show the sequence similarity seen in the various VH andVL regions, which can be present in an antibody according to the presentinvention. The VH sequences within VH group1, and the VL sequenceswithin VL group 1 comprise identical CDRs.

FIG. 4a shows a diagram illustrating Grading of Skin GvHD of patientsenrolled in a study concerned with the administration of CDina26.

FIG. 4b shows a diagram illustrating Grading of liver GvHD of patientsenrolled in a study concerned with the administration of CDina26.

FIG. 4c shows a diagram illustrating Grading of gut GvHD of patientsenrolled in a study concerned with the administration of CDina26.

FIG. 5 shows a diagram illustrating absolute CD4 counts.

FIG. 6 shows a diagram illustrating the projected cumulative incidenceof transplant related mortality of 13 control patients with grade III-IVacute GvHD, treated with steroids, cyclosporine and otherimmunosuppressive drugs, compared with 9 patients treated with steroids,cyclosporine and CDina26.

FIG. 7 shows a diagram illustrating the projected actuarial survival of13 control patients with grade III-IV acute GvHD, treated with steroids,cyclosporine and other immunosuppressive drugs, compared with 9 patientstreated with steroids, cyclosporine and CDina26.

FIG. 8 illustrates the structure of preferred antibodies according tothe present invention. The Figure shows two different groups of lightchain that can form an antibody of the present invention (VL group 1 andVL group 3).

FIG. 9 illustrates Flow cytometry analysis utilized to determine theability of CDina26 to bind to cell surface expressed CD26.

FIG. 10 Summary of SEQ ID NOs. Each one of the VL amino acid sequencescan be present in combination with one of the VH amino acid sequences inan antibody of the present invention, optionally further in combinationwith the CL amino acid sequence having SEQ ID NO: 48 and a CH1-CH2-CH3sequence having SEQ ID NO: 49. FIG. 10 also indicates nucleotidesequences having SEQ ID NOs: 50 to 128, corresponding to amino acidsequences having SEQ ID NOs: 4 to 49.

FIG. 11 shows binding of Human (left) and Porcine (right) Antigen (Ag)on captured CDina26, measured in Biacore® Resonance Units (RU).

FIG. 12 shows a bar plot comparison of CDina26 binding to the 7identified discontinuous binding regions in CD26: DYDESSGRWNCLVAR (SEQID NO: 146); DVTWATQERISLQWL (SEQ ID NO: 147); TTGWVGRFRPSEPHF (SEQ IDNO: 153); TFITKGTWEVIG (SEQ ID NO: 155); DYLYYISNE (SEQ ID NO: 156);SCELNPERCQYY (SEQ ID NO: 157); and SGPGLP (SEQ ID NO: 158).

DETAILED DESCRIPTION OF THE INVENTION

During numerous experiments leading to the present invention, theinventors surprisingly found an antibody which can be used with highlybeneficial and promising results as a medicament, in particular fortreating and/or preventing at least one of Graft-versus-Host Disease(GvHD) and Aplastic Anemia, as well as for promoting engraftment afterhaematopoietic stem cell transplantation. The antibody of the presentinvention can exhibit specific binding to CD26, in particular to humanCD26, especially human CD26 present on a stem cell or expressed on anactivated T lymphocyte. Binding of an antibody of the present inventionto human CD26 expressed on activated T lymphocytes (in particularsubpopulations of CD16+CD3+T and CD56+CD3+T) is a particularlyadvantageous property of an antibody of the present invention, asdiscussed below. The rationale for the use of murine monoclonal antibodyagainst CD26 for treating aGvHD steroid resistant is mainly supported byits ability to block CD26 activity. Experiments performed by the presentinventors show that CD26 is over-expressed in stimulated T cells andover-expressed in lower amount in stimulated Natural Killer cells. Onthe contrary, this molecule is low expressed on resting cells. B cells,monocytes and dendritic cells never express CD26, neither do mesenchymalstem cells, endothelial cells and fibroblasts express CD26. An anti-CD26of the present invention specifically binds to activated regulatory Tcells, interfering with their expansion and with their role in themodulation of the immune response. While not wishing to be bound by anytheory, it is currently assumed that activated lymphocytes are a targetof anti-CD26 and that a partial depletion of activated lymphocytes couldlead to a clinically relevant modulation of at least one of GvHD, inparticular aGvHD, especially steroid resistant aGvHD, of AplasticAnemia, and of disease(s), disorder(s) and/or condition(s) presentbefore and/or during and/or after haematopoietic stem celltransplantation, as well as could promote engraftment afterhaematopoietic stem cell transplantation. While not wishing to be boundby any theory, it is currently assumed that donor T lymphocytes canstill mount a reaction directly against tumour cells.

Haematopoietic stem cell transplantation (HCT) represents a standardtreatment for hematologic diseases and malignancies.

While not wishing to be bound by any theory, it is currently assumedthat inhibition or depletion of CD26 on donor cells by administering oneor more antibodies of the present invention, in particular CDina26, canenhance engraftment, in particular short-term engraftment, as well ascan enhance repopulation, in particular competitive repopulation,secondary transplantation and survival of the treated subject, forexample humans and mice. Furthermore, while not wishing to be bound byany theory, it is currently assumed that if homing and engraftment isenhanced, in particular by administering at least one antibody of thepresent invention, especially CDina26, the time to recovery ofhaematopoietic lineages can be reduced resulting in less engraftmentfailure and better overall survival, especially in hUCB (human umbilicalcord blood cell) transplantation.

An antibody of the present invention for use as a medicament or atherapy using this antibody can provide more patients with best chancesof a successful outcome after haematopoietic cell transplantation.Furthermore, one or more antibodies of the present invention againstCD26 antigen, in particular, CDina26, can give important clinicalbenefit in patients who have undergone haematopoietic stem celltransplantation for at least one of treating steroid resistant acuteGvHD and improving the engraftment that is correlated, directly, withoverall survival.

While not wishing to be bound by any theory, it is currently assumedthat administering at least one antibody of the present invention, inparticular CDina26, can provide a beneficial activity, in particularpromoting engraftment after haematopoietic stem cell transplantationand/or preventing and/or treating at least one of Graft-versus-HostDisease and Aplastic Anemia, through the binding to CD26 as membraneglycoprotein that mediates signaling pathway.

Surprisingly, the present inventors provide one or more antibodies, inparticular CDina26, solving the before-mentioned problems.

According to one aspect, the present invention provides an antibody,which antibody can specifically bind to CD26 glycoprotein. Inparticular, this antibody can specifically bind to human CD26,especially to human CD26 present on stem cell(s) (in particular humanstem cell(s)) and/or to human CD26 expressed on T lymphocytes (inparticular subpopulations of CD16+CD3+T and CD56+CD3+T), especially onactivated T lymphocyte(s) (in particular subpopulations of CD16+CD3+Tand CD56+CD3+T). Unless explicitly indicated otherwise, the terms CD26and CD26 glycoprotein are used interchangeably herein.

This antibody can comprise a heavy chain variable region and a lightchain variable region. The heavy chain variable region of this antibodycan comprise the sequence set forth in SEQ ID NO: 1. The light chainvariable region of this antibody can comprise the sequence set forth inSEQ ID NO: 2 or the sequence set forth in SEQ ID NO: 3 or both thesequence set forth in SEQ ID NO: 2 and the sequence set forth in SEQ IDNO: 3. The heavy chain variable region can comprise a CDR3 comprisingthe sequence set forth in SEQ ID NO: 1. The light chain variable regioncan comprise a CDR3 comprising the sequence set forth in SEQ ID NO: 3 orthe sequence set forth in SEQ ID NO: 2 or both the sequence set forth inSEQ ID NO: 2 and the sequence set forth in SEQ ID NO: 3. The heavy chainvariable region comprising the sequence set forth in SEQ ID NO: 1, inparticular comprising a CDR3 comprising the sequence set forth in SEQ IDNO: 1, can furthermore comprise a CDR1 and a CDR2, wherein the aminoacid sequences of the CDR1 of the heavy chain variable region and theCDR2 of the heavy chain variable region are those of a heavy chainvariable region of an antibody produced by the hybridoma cell linedeposited at CBA-ICLC of Genoa (Italy) as PD 12002, said heavy chainvariable region of an antibody produced by the hybridoma cell linedeposited at CBA-ICLC as PD 12002 comprising SEQ ID NO: 1, in particulara CDR3 comprising SEQ ID NO: 1. The light chain variable regioncomprising the sequence set forth in SEQ ID NO: 2, in particularcomprising a CDR3 comprising the sequence set forth in SEQ ID NO: 2, canfurthermore comprise a CDR1 and a CDR2, wherein the amino acid sequencesof the CDR1 of the light chain variable region and the CDR2 of the lightchain variable region are those of a light chain variable region of anantibody produced by the hybridoma cell line deposited at CBA-ICLC as PD12002, said light chain variable region of an antibody produced by thehybridoma cell line deposited at CBA-ICLC as PD 12002 comprising SEQ IDNO: 2, in particular a CDR3 comprising SEQ ID NO: 2. The light chainvariable region comprising the sequence set forth in SEQ ID NO: 3, inparticular comprising a CDR3 comprising the sequence set forth in SEQ IDNO: 3, can furthermore comprise a CDR1 and a CDR2, wherein the aminoacid sequences of the CDR1 of the light chain variable region and theCDR2 of the light chain variable region are those of a light chainvariable region of an antibody produced by the hybridoma cell linedeposited at CBA-ICLC as PD 12002, said light chain variable region ofan antibody produced by the hybridoma cell line deposited at CBA-ICLC asPD 12002 comprising SEQ ID NO: 3, in particular a CDR3 comprising SEQ IDNO: 3. In particular, the CDR1 and CDR2 of the light chain variableregion can be those of a light chain variable region of an antibodyproduced by the hybridoma cell line deposited at CBA-ICLC as PD 12002and the CDR1 and CDR2 of the heavy chain variable region can be those ofa heavy chain variable region of said antibody produced by saidhybridoma cell line deposited at CBA-ICLC as PD 12002.

Additionally or alternatively, an antibody, which can specifically bindto CD26, can comprise a heavy chain variable region comprising a CDR3(SEQ ID NO: 1) and a light chain variable region comprising a CDR3 (SEQID NO: 2 and/or 3), wherein the amino acid sequence of the CDR3 of theheavy chain variable region is that of a heavy chain variable region ofan antibody produced by the hybridoma cell line deposited at CBA-ICLC ofGenoa (Italy) as PD 12002, and the amino acid sequence of the CDR3 ofthe light chain variable region is that of a light chain variable regionof an antibody produced by the hybridoma cell line deposited as PD12002. In particular, the antibody. which can specifically bind to CD26,can comprise a heavy chain comprising this heavy chain variable regionand a light chain comprising this light chain variable region. CDR3sequences abovementioned are listed in FIG. 2 a.

Additionally or alternatively, an antibody, which can specifically bindto CD26, can comprise a heavy chain variable region comprising a CDR1, aCDR2, and a CDR3 and a light chain variable region comprising a CDR1, aCDR2, and a CDR3, wherein the amino acid sequences of the CDR1, CDR2,and CDR3 of the heavy chain variable region are those of a heavy chainvariable region of an antibody produced by the hybridoma cell linedeposited at CBA-ICLC of Genoa (Italy) as PD 12002, and the amino acidsequences of the CDR1, CDR2, and CDR3 of the light chain variable regionare those of a light chain variable region of an antibody produced bythe hybridoma cell line deposited as PD 12002. In particular, thisantibody which can specifically bind to CD26, can comprise a heavy chaincomprising this heavy chain variable region and a light chain comprisingthis light chain variable region. CDR1, CDR2 and CDR3 sequencesabovementioned are listed in FIG. 2 b.

Additionally or alternatively, the antibody, which can specifically bindto CD26, can comprise a heavy chain variable region and a light chainvariable region, wherein the amino acid sequences of the heavy chainvariable region are those of a heavy chain variable region of anantibody produced by the hybridoma cell line deposited at CBA-ICLC ofGenoa (Italy) as PD 12002, and the amino acid sequences of the lightchain variable region are those of a light chain variable region of thisantibody produced by the hybridoma cell line deposited as PD 12002. Inparticular, the antibody, which can specifically bind to CD26, cancomprise a heavy chain comprising this heavy chain variable region and alight chain comprising this light chain variable region. In particular,the amino acid sequences of the heavy chain can be those of a heavychain of an antibody produced by the hybridoma cell line deposited as PD12002. and/or the amino acid sequences of the light chain can be thoseof a light chain of this antibody produced by the hybridoma cell linedeposited as PD 12002. In particular, the antibody, which canspecifically bind to CD26, can comprise the same heavy chain sequencesand the same light chain sequences as an antibody produced by thehybridoma cell line deposited as PD 12002.

Additionally or alternatively, the antibody, which can specifically bindto CD26, can comprise a heavy chain variable region and a light chainvariable region, wherein the amino acid sequences of the heavy chainvariable region are those of a heavy chain variable region of anantibody produced by the hybridoma cell line deposited at CBA-ICLC ofGenoa (Italy) as PD 12002, and the amino acid sequences of the lightchain variable region are those of a light chain variable region of anantibody produced by the hybridoma cell line deposited as PD 12002. In aspecific embodiment, an antibody of the present invention can comprise aheavy chain variable region comprising sequence ID NO: 26 and a lightchain variable region comprising sequence ID NO: 4 and/or sequence IDNO: 5. In particular, the antibody, which can specifically bind to CD26,can comprise a heavy chain comprising this heavy chain variable regionand a light chain comprising this light chain variable region. Inparticular, the amino acid sequences of the heavy chain are those of aheavy chain of an antibody produced by the hybridoma cell line depositedas PD 12002, and/or the amino acid sequences of the light chain arethose of a light chain of an antibody produced by the hybridoma cellline deposited as PD 12002.

According to a preferred embodiment, the antibody of the presentinvention can bind to the region of amino acid positions 290-550 of thehuman CD26 sequence, referring to the human CD26 sequence as publishedin the prior art.

According to a further embodiment, the antibody of the present inventiondoes not specifically bind to porcine CD26. According to one embodiment,the epitope of an anti-human CD26 antibody of the present inventioncomprises at least one, for example, one, two, three, four, five, ormore of the 358 amino acid residues resulting from the differencebetween human and porcine CD26. Thus, according to this embodiment ofthe invention, the antibody therefore recognizes such different regionbetween human and porcine CD26.

According to one embodiment of the present invention, the antibodymixture of the present invention does not comprise an antibody notspecifically binding to human CD26.

Additionally or alternatively, an antibody, which can specifically bindto CD26, can comprise a heavy chain variable region comprising an ABR1,an ABR2, and an ABR3 and a light chain variable region comprising anABR1, an ABR2, and an ABR3, wherein the amino acid sequences of theABR1, ABR2, and ABR3 of the heavy chain variable region are those of aheavy chain variable region of an antibody produced by the hybridomacell line deposited at CBA-ICLC of Genoa (Italy) as PD 12002, and theamino acid sequences of the ABR1, ABR2, and ABR3 of the light chainvariable region are those of a light chain variable region of anantibody produced by the hybridoma cell line deposited as PD 12002.ABR1, ABR2 and ABR3 sequences abovementioned are listed in FIG. 2 c.

According to one embodiment of the present invention, the followingAntigen Binding Regions (ABRs) are present in the antibody of thepresent invention:

ABR1 (light chain) comprises the amino acid sequence: SSVSYMN (SEQ IDNO: 135),

ABR2 (light chain) comprises the amino acid sequence: LWIYSTSNLAS (SEQID NO: 136),

ABR3 (light chain) comprises the amino acid sequence: QQRSSYPN (SEQ IDNO: 137),

wherein preferably ABR3 is included in SEQ ID NO: 2,

or

ABR1 (light chain) comprises the amino acid sequence: ENVVTYVS (ABR1*)(SEQ ID NO: 138),

ABR2 (light chain) comprises the amino acid sequence: LLIYGASNRYT(ABR2*) (SEQ ID NO: 139),

ABR3 (light chain) comprises the amino acid sequence: GQGYSYPY (ABR3*)(SEQ ID NO: 140), wherein preferably ABR3 is included in SEQ ID NO: 3.

According to a further embodiment of the present invention, thesequences ABR1 to 3 or ABR1* to 3* are present in an antibody of theinvention together with the following Antigen Binding Regions (ABRs):

ABR1 (heavy chain) comprising the amino acid sequence: YTFRSYDIN (ABR1h)(SEQ ID NO: 141),

ABR2 (heavy chain) comprising the amino acid sequence: WIGWIFPGDGSTKY(ABR2h) (SEQ ID NO: 142),

ABR3 (heavy chain) comprising the amino acid sequence: RWTVVGPGYFDV(ABR3h) (SEQ ID NO: 143),

wherein preferably ABR3 (heavy chain) is included in SEQ ID NO: 1.

According to one embodiment, the ABRs are determined according to the“Paratome tool” as published in Kunik V, Peters B, Ofran Y (2012)“Structural Consensus among Antibodies Defines the Antigen BindingSite”, PLoS Comput Biol 8(2): e1002388.doi:10.1371/journal.pcbi.1002388;Editor Brian Baker, University of Notre Dame, United States of America;Published Feb. 23, 2012; see alsohttp://ofranservices.biu.ac.il/site/services/paratome/index.html.V_(H)/V_(L) ABR sequences abovementioned are listed in FIG. 2 b.

The term “antibody” as used in the context of the present applicationcan encompass whole antibody molecules, full-length immunoglobulinmolecules, in particular naturally occurring full-length immunoglobulinmolecules or full-length immunoglobulin molecules formed byimmunoglobulin gene fragment recombinatorial processes, as well asantibody fragments. Antibody fragments can be in particular antibodyfragments comprising at least one antibody-antigen binding site.Antibody fragments can in particular exhibit specific binding to CD26,in particular human CD26, which can be for example present on a stemcell (in particular human stem cell) and/or expressed on T lymphocyte(s)(in particular subpopulations of CD16+CD3+T and CD56+CD3+T), inparticular on activated T lymphocyte(s). Furthermore, the term“antibody” as used in the context of the present application canencompass fusion proteins, in particular exhibiting specific binding toCD26, especially human CD26, which can be present on a stem cell and/orexpressed on T lymphocyte(s), in particular on activated Tlymphocyte(s). An antibody-antigen binding site can be in particular anantigen binding site of an antibody comprising at least one CDRsequence.

The term “antibody” can include e.g. monoclonal, polyclonal,multispecific (for example bispecific), recombinant, human, chimeric andhumanized antibodies. Furthermore, the term “antibody” can alsoencompass recombinantly expressed antigen binding proteins and antigenbinding synthetic peptides. In particular, the term “antibody” can e.g.encompass minibodies, and diabodies, all of which preferably can exhibitspecific binding to CD26, especially human CD26. Furthermore, the term“antibody”, as used herein, can encompass immunoglobulins produced invivo, as well as those produced in vitro, in particular by a hybridoma.Moreover, the terms “antibody” or “at least one antibody” can encompassantibody mixtures. The term “antibody mixture” in particular encompassesa mixture comprising or consisting of two or more antibodies exhibitingspecific binding to CD26, especially human CD26, in particularcomprising at least one antibody of the present invention. The two ormore antibodies present in the mixture can be two or more differentantibodies, for example two or more antibodies which do not haveidentical amino acid sequences. In particular, an antibody differentfrom another antibody can be an antibody having an amino acid sequence,wherein at least one amino acid residue has been deleted, inserted orreplaced with a different amino acid residue, compared to the amino acidsequence of said another antibody. A particularly useful antibodymixture according to the invention comprises or consists of theantibodies produced by the hybridoma cell line deposited as PD 12002, orcomprises at least one of the antibodies produced by the hybridoma cellline deposited as PD 12002.

An antibody according to present invention can be a recombinantlyproduced antibody. An antibody of the present invention can be amonoclonal and/or murine antibody, in particular a murine monoclonalantibody. As mentioned above, at least one antibody of the presentinvention can be an antibody mixture comprising at least one antibody ofthe present invention, in particular comprising or consisting of theantibodies produced by the PD 12002 hybridoma deposit, CDina26.

The term “monoclonal antibody” refers to a substantially homogeneousantibody population involved in the highly specific recognition andbinding of a single antigenic determinant, or epitope. This is incontrast to polyclonal antibodies that typically include differentantibodies directed against different antigenic determinants. The term“monoclonal antibody” encompasses both intact and full-length monoclonalantibodies as well as antibody fragments (such as Fab, Fab′, F(ab′)2,Fv), single chain (scFv) mutants, fusion proteins comprising an antibodyportion, and any other modified immunoglobulin molecule comprising anantigen recognition site. Furthermore, “monoclonal antibody” refers tosuch antibodies made in any number of manners including but not limitedto by hybridoma, phage selection, recombinant expression, and transgenicanimals.

The term “humanized antibody” refers to forms of nonhuman (e.g. murine)antibodies that are specific immunoglobulin chains, chimericimmunoglobulins, or fragments thereof that contain minimal non-human(e.g. murine) sequences. Typically, humanized antibodies are humanimmunoglobulins in which residues from the complementary determiningregion (CDR) are replaced by residues from the CDR of a non-humanspecies (e. g. mouse, rat, rabbit, and hamster) that have the desiredspecificity, affinity, and capability (Jones et al., 1986, Nature,321:522-525; Riechmann et al., 1988, Nature, 332:323-327; Verhoeyen etal., 1988, Science, 239: 1534-1536). In some instances, the Fv frameworkregion (FR) residues of a human immunoglobulin are replaced with thecorresponding residues in an antibody from a non-human species that hasthe desired specificity, affinity, and capability. The humanizedantibody can be further modified by the substitution of additionalresidues either in the Fv framework region and/or within the replacednon-human residues to refine and optimize antibody specificity,affinity, and/or capability. In general, the humanized antibody willcomprise substantially all of at least one, and typically two or three,variable domains containing all or substantially all of the CDR regionsthat correspond to the non-human immunoglobulin whereas all orsubstantially all of the FR regions are those of a human immunoglobulinconsensus sequence. The humanized antibody can also comprise at least aportion of an immunoglobulin constant region or domain (Fc), typicallythat of a human immunoglobulin.

The term “chimeric antibodies” refers to antibodies wherein the aminoacid sequence of the immunoglobulin molecule is derived from two or morespecies. Typically, the variable region of both light and heavy chainscorresponds to the variable region of antibodies derived from onespecies of 60 mammals (e.g. mouse, rat, rabbit, etc) with the desiredspecificity, affinity, and capability while the constant regions arehomologous to the sequences in antibodies derived from another (usuallyhuman) to avoid eliciting an immune response in these species. Typicallychimeric antibodies utilize rodent variable regions (VH and VL) andhuman constant regions, in order to produce an antibody withpredominantly human domains. The production of such chimeric antibodiesis well known in the art, and may be achieved by standard means.Sequences of human constant regions will be apparent to the skilledperson and/or are available in public databases (e.g. National centerfor Biotechnology Information (NCBI), U.S. National Library ofMedicine).

The term “antibody fragment” can refer to a fragment, such as F(ab′)₂,Fab, F(ab)₂, Fab′, Fv, dAb, scFv, heavy chain variable region CDR1,heavy chain variable region CDR2, heavy chain variable region CDR3,light chain variable region CDR1, light chain variable region CDR2,light chain variable region CDR3, single chain variable fragment (scFv),VH, VL, and the like, all of which preferably can exhibit specificbinding to CD26, especially human CD26. An “antibody fragment” canspecifically bind with the same antigen that is recognized by the wholeantibody or full-length antibody. An “antibody fragment” can be inparticular a portion of an intact antibody.

Antibody fragments which recognize specific epitopes, in particularwhich specifically bind to CD26, can be generated by a skilled personapplying techniques known in the art. Fragments of an antibody, inparticular fragments of an antibody, which can specifically bind toCD26, especially human CD26, such as e.g. fragments of one or moreanti-CD26 antibodies produced by the PD 12002 hybridoma deposit,CDina26, can be e.g. prepared by enzymatically treating the antibody toobtain antibody fragments. Furthermore, an antibody fragment can beproduced by expression of DNA coding for the fragment in a host, such ase.g. E. coli, B. subtilis, P. pastoris, K. lactis. An antibody fragmentcan be e.g. prepared by proteolytic hydrolysis of a full lengthantibody. Enzymes, in particular proteolytic enzymes, for obtainingantibody fragments are known to a skilled person and include, but arenot limited to, e.g. papain, pepsin and/or plasmin. In particular, anantibody fragment can be e.g. prepared by pepsin or papain digestion offull length antibodies by applying procedures known to a skilled person,as mentioned e.g. in US 2010/0196266 A1. Such procedures are described,for example, in Goldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647, aswell as in the references cited therein. Procedures for preparingantibody fragments are known in the art and are described e.g. inNisonoff et al., Arch Biochem. Biophys. 89: 230 (1960); Porter, Biochem.J. 73: 119 (1959), Edelman, METHODS IN ENZYMOLOGY VOL. 1, page 422(Academic Press 1967), and Coligan et al., CURRENT PROTOCOLS INIMMUNOLOGY, VOL. 1, (John Wiley & Sons 1991), p. 2.8.1-2.8.10 and2.10.-2.10.4.

As used herein, the term “heavy chain” includes a full-length heavychain and fragments thereof, which are preferably capable ofspecifically binding to CD26, especially human CD26. A full-length heavychain can include a heavy chain variable region, VH, and three regions,CH1, CH2, and CH3.

As used herein, the term “light chain” can in particular refer to afull-length light chain and fragments thereof, which preferably arecapable of specifically binding to CD26, especially human CD26. Afull-length light chain can comprise a light chain variable region, VL,and a light chain constant region, CL.

As used herein, the term “variable region” of an antibody can refer to avariable region of the antibody light chain or to a variable region ofthe antibody heavy chain or to a combination of the before-mentionedvariable regions. The variable regions of the light and heavy chain caneach comprise four framework regions (FR) connected by threecomplementarity determining regions (CDRs). Two definitions of CDRlocation are currently in use in the art. The first one is the “sequencevariability” definition of Kabat et al. (“Sequences of Proteins ofImmunological Interest”, 4^(th) ed., Washington, D.C., Public HealthService, N.I.H., which is incorporated herewith by reference). Accordingto a preferred embodiment, the definition of Kabat et al. is used in thepresent application. Alternatively, the CDR regions can also be definedusing the structural variability definition of Chothia and Lesk (Chothiaet al., J. Mol. Biol. 1987, 196(4):901-17, which is incorporatedherewith by reference).

As used herein, the term “constant region” of an antibody refers to aconstant region of the antibody light chain or a constant region of theantibody heavy chain or to a combination of before-mentioned constantregions.

For producing antibodies, in particular human, humanized, chimericantibodies, and fragments thereof, for example any of the methods asdisclosed in US 2010/0196266 A1, which document is incorporated hereinby reference, can be used.

Antibody fragments can be produced by several methods including, but notlimited to, the following methods, such as e.g. described in US2010/0196266 A1:

F(ab′)₂ fragments can be generated by pepsin digestion of the antibodymolecule. Fab′ fragments can be for example obtained by reducingdisulfide bridge(s) of the F(ab′)₂ fragments. Alternatively, Fab′expression libraries can be for example constructed as described e.g. byHuse et al. (Science 1989, 246:1274-1281). Fab′ expression librariesallow an identification of monoclonal Fab′ fragments having aspecificity of interest, in particular of fragments binding to CD26.

F(ab)₂ fragments can be produced by papain digestion of an antibody. Fabfragments can be obtained by disulfide reduction. A “Fab fragment”represents in particular a fragment that is comprised of one light chainand the CH1 and variable regions of one heavy chain. The heavy chain ofa Fab molecule cannot bind via a disulfide bond to another heavy chainmolecule.

Furthermore, an antibody fragment can be also a single variable regionor a peptide consisting of or comprising a singlecomplementarity-determining region (CDR).

Moreover, the antibody of the present invention can be a diabody. Asused herein, “diabodies” can describe in particular small antibodyfragments with two antigen-binding sites, which fragments comprise aheavy chain variable domain (VH) connected to a light chain variabledomain (VL) in the same polypeptide chain (VH-VL). Unless explicitlymentioned to the contrary, the terms variable domain and variable regionare used herein interchangeably. Diabodies and techniques for theirproduction are discussed for example in EP 404 097, WO 93/11161, and inHollinger et al., 1993, Proc. Natl. Acad Sci. USA 90: 6444-6448.

Furthermore, the antibody of the present invention can be a single chainFv molecule. A single chain Fv molecule (abbreviated as scFv) comprisesa VL domain and a VH domain, which can associate to form a binding site,in particular for CD26. These two domains are further covalently linkedby a peptide linker, such as e.g. by a peptide comprising 1 to 25 aminoacid residues. Unless explicitly mentioned to the contrary, the terms VLdomain and VL region are used herein interchangeably. Moreover, unlessexplicitly mentioned to the contrary, the terms VH domain and VH regionare used herein interchangeably. Methods for obtaining scFv moleculesare for example described in U.S. Pat. No. 4,704,692, U.S. Pat. No.4,946,778, R. Raag and M. Whitlow, “Single Chain Fvs.” FASEB Vol.9:73-80 (1995) and R. E. Bird and B. W. Walker, “Single Chain AntibodyVariable Regions,” TIBTECH, Vol. 9:132-137 (1991).

Furthermore, the term antibody as used herein also encompasses singledomain antibodies. Methods for preparing single domain antibodies (DABs)are known to a skilled person and are for example described in Cossinset al. (2006, Prot Express Purif 51:253-259), incorporated herein byreference.

According to one embodiment, an antibody or fragment thereof accordingto the present invention can contain at least a heavy chain CDR3, and atleast a light chain CDR3; in particular, an antibody or fragment thereofaccording to the present invention can contain the sequence set forth inSEQ ID NO: 1, as well as at least one of the sequences set forth in SEQID NO: 2 and 3.

Antibody fragments can comprise at least 4 amino acids, at least 5 aminoacids, at least 7 amino acids, at least 9 amino acids, in particular atleast 15 amino acids. An antibody fragment of the present invention canhave any upper size limit, and can have for example merely one aminoacid residue less than the full-length antibody from which it isobtained.

The antibody of the present invention can be a bispecific antibody,which is capable of binding to CD26, in particular human CD26.Bispecific antibodies can be produced by several methods including e.g.fusion of hybridomas or linking of Fab′ fragments. Such methods aredescribed e. g. in Songsivilai et al., 1990, Clin. Exp. Immunol. 79:315-321; Kostelny et al., 1992, J. Immunol. 148: 1547-1553.

According to an embodiment, the antibody of the present invention can bea monoclonal antibody. Methods for preparing monoclonal antibodiesagainst a target antigen are known in the art, as may be seen forexample from Coligan et al. (eds.), CURRENT PROTOCOLS IN IMMUNOLOGY,VOL. 1, pages 2.5.1-2.6.7 (John Wiley & Sons 1991), Kohler and Milstein,Nature 256: 495 (1975), and US 2010/0196266 A1. Monoclonal antibodiesare for example obtainable by methods known to a skilled person, such asdisclosed in US 2010/0196266 A1. In particular, monoclonal antibodiesare obtainable by methods comprising one or more, preferably all, of thefollowing steps: injecting mammal(s), for example a mouse with acomposition comprising an antigen, removing spleen from these injectedmammal(s) to obtain B-lymphocytes, fusing the so obtained B-lymphocyteswith myeloma cells to produce hybridomas, cloning the hybridomas,selecting at least one positive clone producing antibodies to theantigen, culturing the at least one positive clone producing antibodiesto the antigen, and isolating the antibodies from the hybridomacultures.

MAbs (monoclonal antibodies) can be isolated and purified from hybridomacultures using known procedures, such as disclosed in US 2010/0196266A1. In particular, one or more isolation and/or purification proceduresselected from the group consisting of size-exclusion chromatography,affinity chromatography, in particular with Protein-A Sepharose, andion-exchange chromatography can be applied. Isolation and/orpurification techniques for antibodies are for example disclosed inBaines et al., “Purification of Immunoglobulin G (IgG),” in METHODS INMOLECULAR BIOLOGY, VOL. 10, pages 79-104 (The Humana Press, Inc. 1992),as well as in Coligan et al., supra, pages 2.7.1-2.7.12 and pages2.9.1-2.9.3.

The term “monoclonal antibody” can in particular describe an antibodyobtained from a population of substantially homogeneous antibodies,wherein the individual antibodies are identical except for possiblenaturally occurring mutations that can be present in low amounts.

After the initial raising of antibodies to the immunogen, in particularafter the initial raising of antibodies that can specifically bind toCD26, the antibodies can be sequenced and then produced usingrecombinant techniques. Humanization and chimerization of non-humanmammal (e.g. murine) antibodies and antibody fragments are well known tothe skilled person.

The antibody of the present invention can be a humanized antibody, inparticular a humanized monoclonal antibody. The term “humanizedantibody” can in particular encompass antibodies produced by recombinantDNA techniques, in which some or all of the amino acids of a humanimmunoglobulin light or heavy chain that are not required for antigenbinding (such as e.g. some or all of the amino acids of constant regionsand framework regions of variable domains) are used to substitute forthe corresponding amino acids from the light or heavy chain of thenon-human mammal antibody (e.g. murine) antibody. Methods for producinghumanized monoclonal antibodies are known in the art and are describedfor example in the following publications: Jones et al., Nature 321: 522(1986), Carter et al., Proc. Nat'l Acad. Sci. USA 89: 4285 (1992),Riechmann et al., Nature 332: 323 (1988), Verhoeyen et al., Science 239:1534 (1988), Sandhu, Crit. Rev. Biotech. 12: 437 (1992), and Singer etal., J. Immun. 150: 2844 (1993). An antibody, such as e.g. a chimeric ornon-human mammal (e.g. murine) monoclonal antibody, in particular achimeric or non-human mammalian (e.g. murine) monoclonal antibody of thepresent invention, can be humanized by transferring the non-human mammal(e.g. mouse) CDRs from the light and heavy variable chains of thenon-human mammal immunoglobulin, for example mouse immunoglobulin, intothe corresponding variable domains of a human antibody, as describede.g. in US 2010/0196266 A1. The non-human mammal framework regions (FR),for example mouse framework regions (FR), in the chimeric monoclonalantibody can be also replaced with human FR sequences. For example, anantibody of the present invention which is a humanized version of anon-human mammal (e.g. murine) antibody to CD26 can have on both of itsheavy and light chains constant regions of a human antibody and/orframework regions from the variable domains of a human antibody, and/orCDRs from the non-human mammal (e.g. murine) antibody.

For improving the antibody affinity of a humanized antibody, inparticular for improving its capability of binding to CD26, additionalmodification steps can be carried out, as described e.g. in US2010/0196266 A1. In particular, one or more amino acid residues in thehuman FR regions can be replaced with amino acid residues present atcorresponding positions in the non-human, in particular murine, antibodyin order to maintain or improve the binding affinity of the humanizedantibody to the antigen. Methods which can be applied by a skilledperson are for example described in Tempest et al., Biotechnology 9:266(1991), and Verhoeyen et al., Science 239: 1534 (1988). For example,human FR (framework region) amino acid residues which differ from theirnon-human mammal counterparts, for example murine counterparts, and arelocated close to or directly adjacent to one or more CDR amino acidresidues can represent candidates for substitution.

The antibody of the present invention can be a human antibody. The term“human antibody” can in particular encompass an antibody, which has anamino acid sequence corresponding to that of an antibody produced by ahuman and/or has been made using known techniques for producing humanantibodies. In particular, the term “human antibody” can includeantibodies comprising at least one human heavy chain polypeptide or atleast one human light chain polypeptide.

In particular, the antibody of the present invention can be a fullyhuman antibody. In the context of the present application, the term“fully human antibody” can in particular refer to an antibody containinghuman heavy chain and human light chain polypeptides. Methods forproducing human antibodies, in particular fully human antibodies, usingfor example combinatorial approaches or transgenic animals transformedwith human immunoglobulin loci, are known to a skilled person, as may beseen e.g. from US 2010/0196266 A1. Such methods are for exampledescribed in Conrad and Scheller, 2005, Comb. Chem. High ThroughputScreen. 8:117-26; Mancini et al., 2004, New Microbiol. 27:315-28; Brekkeand Loset, 2003, Curr. Opin. Pharmacol. 3:544-50). A fully humanantibody is for example also obtainable using genetic or chromosomaltransfection methods or using phage display technology. Genetic orchromosomal transfection methods, as well as phage display technologyare known in the art and are described for example in McCafferty et al.,1990, Nature 348:552-553. In particular, human antibodies can be alsoobtained by introducing human immunoglobulin loci into transgenicanimals, such as e.g. mice, goats or cows, wherein the endogenousimmunoglobulin genes were completely or partially inactivated. Suchprocedures are described e.g. in U.S. Pat. No. 5,545,806, U.S. Pat. No.5,633,425, and U.S. Pat. No. 5,661,016. According to an alternativeprocedure, the human antibody can be obtained by immortalizing human Blymphocytes that produce an antibody directed against a target antigen,in particular that produce an antibody to CD26. Such procedures areknown in the art and are described e.g. in Cole et al., MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boemer etal., 1991, J. Immunol., 147 (I):86-95.

In particular, the phage display technique can be used for generating ahuman antibody, as known in the art and as described e.g. inDantas-Barbosa et al., 2005, Genet. Mol. Res. 4:126-40 and US2010/0196266 A1. Human antibodies can be generated from normal humans orfrom humans having a particular disease state (Dantas-Barbosa et al.,2005). This technique allows constructing human antibodies from adiseased individual.

For example, a phage display library of human Fab antibody fragmentsfrom osteosarcoma patients can be constructed, as disclosed inDantas-Barbosa et al. (2005, supra) and as discussed e.g. in US2010/0196266 A1. In particular, total RNA can be obtained fromcirculating blood lymphocytes (Id.). Recombinant Fab can be cloned fromp, y and K chain antibody repertoires and inserted into a phage displaylibrary (Id.). RNAs can be converted to cDNAs and used to provide FabcDNA libraries using specific primers against heavy and light chainimmunoglobulin sequences (Marks et al., 1991, J. Mol. Biol. 222:581-97).In a next step, library construction can be performed as known to askilled person and as described for example by Andris-Widhopf et al.(2000), Phage Display Laboratory Manual, 1^(st) edition, Cold SpringHarbor Laboratory Press, pp. 9.1 to 9.22). The final Fab fragments canbe digested with restriction endonucleases and inserted into abacteriophage genome to produce the phage display library. Finally, theso obtained libraries can be screened using standard phage displaymethods, as known in the art and as described e.g. in Pasqualini andRuoslahti, 1996, Nature 380:364-366; Pasqualini, 1999, The Quart. J.Nucl. Med. 43:159-162). Phage display can be performed in severalformats, as may be seen e.g. from Johnson and Chiswell, 1993, CurrentOpinion in Structural Biology 3:5564-571.

Moreover, human antibodies can be generated by in vitro activated Bcells. This procedure is described e.g. in U.S. Pat. No. 5,567,610 andU.S. Pat. No. 5,229,275, which both are incorporated herein byreference.

The antibody of the present invention can be a chimeric antibody. Inparticular, a chimeric antibody can be a recombinant protein, whereinthe variable regions of a human antibody have been replaced by thevariable regions of a non-human mammal antibody, such as e.g. a mouseantibody or a rabbit antibody, including the complementarity-determiningregions (CDRs) of the non-human mammal antibody, e.g. the mouse antibodyor rabbit antibody. Procedures for cloning non-human mammalimmunoglobulin variable domains, in particular murine immunoglobulinvariable domains, are known in the art and are for example described byOrlandi et al., Proc. Nat'l Acad. Sci. USA 86: 3833 (1989), and US2010/0196266 A1. Methods for obtaining chimeric antibodies are known tothe skilled person, as may be e.g. seen from Leung et al., Hybridoma13:469 (1994), wherein the production of an LL2 chimera is described.

Antibodies of the present invention can furthermore comprise one or moreadditional moieties to effect desired functions. In particular, theantibodies can include one or more toxin moieties (such as e. g. atetanus toxoid) or radionuclide(s), and/or one or more moieties (such ase.g. biotin, fluorescent moiety, radioactive moiety, histidine tag orother peptide tags) for facilitating isolation and/or detection and/ortargeting, wherein said tag preferably does not or does essentially notalter the binding specificity of said antibody.

The terms “has specificity for”, “exhibits a specific binding to”,“capable of specifically binding to” and “specifically binds to” areused interchangeably in the present application and can in particularindicate that the antibody reacts or associates more frequently, morerapidly, with greater duration, with greater affinity, or with somecombination of the above to an epitope or protein than with alternativesubstances, including unrelated proteins. According to one embodiment,“binding” and “specifically binding”, as well as “antibody binding to”and “antibody specifically binding to” may be used interchangeably inthe context of the present invention.

In certain embodiments, an anti-CD26 disclosed herein binds to humanCD26 with a kinetic dissociation rate (K_(off)) of about 1·e⁻³ to 1·e⁻⁵s⁻¹, preferably 5·e⁻³ to 5·e⁻⁴ s⁻¹, more preferably 8·e⁻³ to 3·e⁻⁴ s⁻¹in particular about 1.32·e⁻⁴ s⁻¹.

In certain embodiments, an anti-CD26 disclosed herein binds to humanCD26 with a kinetic dissociation constant (K_(D)) Of about 5·e⁻⁸ to5·e⁻¹⁰ M, preferably 2·e⁻⁹ to 1·e⁻¹⁰ M, more preferably 3·e⁻⁹ to 7·e⁻⁹M, in particular about 5.02·e⁻⁹ M.

In certain embodiments, an anti-CD26 disclosed herein binds to humanCD26 with a kinetic association constant (K_(on)) of about 5·e³ to 1·e⁵1/Ms, preferably 1·e⁴ to 5·e⁴ 1/Ms, more preferably 1.5·e⁴ to 3.5·e⁴1/Ms, in particular about 2.63·e⁴ 1/Ms.

In certain embodiments, an anti-CD26 antibody disclosed herein binds tohuman CD26 with a dissociation constant of about 1 nM or less, about 3nM or less, about 6 nM or less, about 12 nM or less, about 30 nM orless, about 60 nM or less, about 200 nM or less.

In some embodiments an anti-CD26 antibody disclosed herein binds tohuman CD26 with a dissociation constant of about 0.1 nM to about 10 nM,about 0.1 nM to about 6 nM, about 0.1 nM to about 3 nM, or about 0.1 nMto about 1 nM.

The antibodies of the present invention can be assayed for specificbinding by any method known to a skilled person, including, but notlimited to, competitive and non-competitive assay systems usingtechniques such as Biacore® analysis, FACS analysis, immunofluorescence,immunocytochemistry, Western blots, radioimmunoassays, ELISA, “sandwich”immunoassays, immunoprecipitation assays, precipitation reactions,immunodiffusion assays, agglutination assays, complement-fixationassays, immunoradiometric assays, fluorescent immunoassays, and proteinA immunoassays. Such assays are described e.g. in Ausubel et al., eds.,1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons,Inc., New York and U.S. Pat. No. 7,982,013 B2, which are incorporated byreference herein in entirety. Preferably, Biacore® analysis may becarried out.

Native antibodies can be made up of two or more heterodimeric subunitseach containing one heavy (H) and one light (L) chain. An individualnative antibody can have one type of L chain and one type of H chain,which are held together by disulfide bonds to form a heterodimericsubunit.

The term “peptide” can in particular refer to a compound that includestwo or more amino acids. The amino acids can be linked together by apeptide bond. A peptide can comprise naturally occurring amino acidsand/or non-naturally occurring amino acids; in particular a peptide cancomprise L-amino acids and/or D-amino acids. Short peptides, e.g.,peptides having less than ten amino acid units, are sometimes referredto as “oligopeptides”. Other peptides having a large number of aminoacid residues, e.g. up to 100 or more, can be referred to as“polypeptides”. As used herein, the term “polypeptide” can refer to anypeptide containing three or more amino acids. As used herein, anyreference to a “polypeptide” also includes an oligopeptide, and anyreference to a “peptide” includes polypeptides, oligopeptides, andproteins.

An antibody of the present invention can be an antibody of any class. Inparticular, an antibody of the present invention can have an antibodyisotype selected from the group consisting of IgG1, IgG2, IgG3, IgG4,IgM, IgA, IgD and IgE. In particular an antibody of the presentinvention can be of IgG2 class, especially of IgG 2B class. The term“isotype”, as used herein, can in particular refer to the antibody class(such as e.g. IgG) that is encoded by heavy chain constant region genes.Sequences of human immunoglobulin constant regions will be apparent tothe skilled person and/or are available in public databases (e.g.National Center for Biotechnology Information (NCBI), U.S. NationalLibrary of Medicine).

Furthermore, an antibody of the present invention specifically bindsCD26 and can have a light chain variable region, which light chainvariable region comprises a variant of a VL CDR1, VL CDR2, or VL CDR3 ofthe light chain variable region of an antibody produced by the hybridomacell line deposited as PD 12002. Moreover, an antibody of the presentinvention specifically binds CD26 and can have a heavy chain variableregion, which heavy chain variable region comprises a variant of a VHCDR1, VH CDR2, or VH CDR3 of the heavy chain variable region of anantibody produced by the hybridoma cell line deposited as PD 12002. Inone embodiment, a variant VH or VL CDR can have at least 90%, preferablyat least 98%, more preferably at least 99% sequence identity to thecorresponding VH or VL CDR of an antibody produced by the hybridoma cellline deposited as PD 12002. Alternatively, a variant VH or VL CDR can bea VH or VL CDR of an antibody produced by the hybridoma cell linedeposited as PD 12002, wherein not more than 5, 4, 3, 2, more preferably1 amino acid residue(s), respectively, have been deleted, inserted orreplaced by an amino acid residue different from the replaced amino acidresidue. In one embodiment, the amino acid replacement is a conservativechange. In one embodiment, the VH and VL CDRs of an antibody produced bythe hybridoma cell line deposited as PD 12002 are listed in FIG. 2 b.

Furthermore, an antibody of the present invention specifically bindsCD26 and can have a light chain variable region, which light chainvariable region comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 4, 5, 6 to 21, a variant of the amino acidsequence of SEQ ID NO: 4, and a variant of the amino acid sequence ofSEQ ID NO: 5. Moreover, an antibody of the present inventionspecifically binds CD26 and can have a light chain variable region,which light chain variable region comprises an amino acid sequenceselected from variants of an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 6 to 21. The variant of SEQ ID NO: 4 can haveat least 90%. preferably at least 98%, more preferably at least 99%sequence identity to SEQ ID NO: 4. The variant of SEQ ID NO: 5 can haveat least 90%, preferably at least 98%, more preferably at least 99%sequence identity to SEQ ID NO: 5. A variant of an amino acid sequenceselected from the group consisting of SEQ ID NOs: 6 to 21 can have atleast 90%, preferably at least 98%, more preferably at least 99%sequence identity to the amino acid sequence selected from the groupconsisting of SEQ ID NOs: 6 to 21. Alternatively, a variant of an aminoacid sequence selected from the group consisting of SEQ ID NOs: 4, 5, 6to 21 can be an amino acid sequence selected from the group consistingof SEQ ID NOs: 4, 5, 6 to 21, wherein not more than 8, preferably notmore than 5, more preferably 1 amino acid residue(s), respectively, havebeen deleted, inserted or replaced by an amino acid residue differentfrom the replaced amino acid residue. In one embodiment, the amino acidreplacement is a conservative change. In particular, an anti-CD26antibody of the present invention can be an antibody, wherein the lightchain variable region can comprise or consist of an amino acid sequenceselected from the group consisting of SEQ ID NOs: 4 to 21 or variantsthereof, as shown e.g. in FIG. 3. Furthermore, an antibody of thepresent invention specifically binds CD26 and can have a light chainvariable region, which light chain variable region comprises amino acidresidues 8-104 of SEQ ID NO: 4 or 5.

A “conservative amino acid change” is a change, wherein one amino acidresidue is replaced with another amino acid residue having a similarside chain. The term is interchangeably used with “conservative aminoacid substitution” or “conservative amino acid variation”. Families ofamino acid residues having similar side chains are known in the art,including basic side chains, acidic side chains, uncharged polar sidechains, nonpolar side chains, beta-branched side chains and aromaticside chains, as discussed e.g. in U.S. Pat. No. 7,982,013 B2, inparticular column 22 thereof. For example, substitution of aphenylalanine for a tyrosine is a conservative substitution. Preferably,the antibody obtained after conservative substitution specifically bindsto CD26, in particular human CD26. Methods for identifying nucleotideand amino acid conservative substitutions which do not eliminate antigenbinding are known in the art (see, e.g., Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999);and Burks et al., Proc. Natl. Acad. Sci. USA 94:412-417 (1997)).

According to one embodiment, in variants of amino acid sequencescomprising one or more CDR sequences, all CDR sequences or at least allCDR3 sequence(s) can remain unchanged. In particular, in variants ofamino acid sequences comprising one or more amino acid sequences setforth in SEQ ID NOs: 1, 2 and 3, the one or more amino acid sequencesset forth in SEQ ID NOs: 1, 2 and 3 can remain unchanged. According toone embodiment, in variants of nucleotide sequences comprising sectionscoding for one or more CDR sequences, all sections coding for CDRsequences or at least all nucleotide sequence sections coding for CDR3sequence(s) can remain unchanged. In particular, in variants ofnucleotide sequences comprising one or more sequences coding for one ormore sequences set forth in SEQ ID NOs: 1, 2 and 3, at least nucleotidesequence sections coding for one or more of SEQ ID NOs: 1, 2 and 3 canremain unchanged. According to one embodiment, in an antibody comprisinga variant of an amino acid sequence selected from the group consistingof SEQ ID NOs: 4 to 21, the light chain variable region can compriseamino acid residues 8-104 of SEQ ID NO: 4 or 5 and/or in an antibodycomprising a variant of an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 22 to 47, the heavy chain variable region cancomprise amino acid residues 7-112 of SEQ ID NO: 26.

“Percent (%) amino acid sequence identity” with respect to a polypeptidesequence as set forth herein is defined as the percentage of amino acidresidues in a candidate sequence of interest to be compared that areidentical with the amino acid residues in a particular polypeptidesequence as set forth herein (e.g. a particular polypeptide sequencecharacterized by a SEQ. ID. NO. in the sequence listings), afteraligning the sequences and introducing gaps, if necessary, to achievethe maximum percent sequence identity, and not considering anyconservative substitutions as part of the sequence identity. A sequencealignment performed for determining percent amino acid sequence identitycan be carried out according to procedures known in the art, asdescribed for example in EP 1 241 179 B1, which is incorporated herewithby reference, including in particular page 9, line 35 to page 10, line40 with the definitions used therein and Table 1 regarding possibleconservative substitutions. For example, a skilled person can usepublicly available computer software. Computer program methods fordetermining sequence identity include, but are not limited to BLAST,BLAST-2, ALIGN or Megalign (DNASTAR) software. According to onepreferred embodiment, the software alignment program used can be BLAST.A skilled person can determine appropriate parameters for measuringalignment, including any algorithms needed to achieve maximal alignmentover the full length of the sequences subjected to comparison. Accordingto a preferred embodiment, the % identity values can be generated usingthe WU-BLAST-2 computer program (Altschul et al., 1996, Methods inEnzymology 266:460-480, which is incorporated herewith by reference), asdescribed e.g. in EP 1 241 179 B1. According to a preferred embodiment,the following parameters are used, when carrying out the WU-BLAST-2computer program: Most of the WU-BLAST-2 search parameters were set tothe default values. The adjustable parameters were set with thefollowing values: overlap span=1, overlap fraction=0.125, word threshold(T)=11, and scoring matrix=BLOSUM62. The HSP S and HSP S2 parameters,which are dynamic values used by BLAST-2, are established by the programitself depending upon the composition of the sequence of interest andcomposition of the database against which the sequence is beingsearched. However, the values can be adjusted to increase sensitivity. A% sequence identity value can be determined by dividing (a) the numberof matching identical amino acid residues between a particular aminoacid sequence as set forth herein which is subjected to comparison (e.g.a particular polypeptide sequence characterized by a SEQ. ID. NO. in thesequence listings) and the candidate amino acid sequence of interest tobe compared, for example the number of matching identical amino acidresidues as determined by WU-BLAST-2, by (b) the total number of aminoacid residues of the polypeptide sequence as set forth herein which issubjected to comparison (e.g. a particular polypeptide sequencecharacterized by a SEQ. ID. NO. in the sequence listings).

“Percent (%) nucleic acid sequence identity” with respect to a nucleicacid sequence as set forth herein is defined as the percentage ofnucleotides in a candidate sequence of interest to be compared that areidentical with the nucleotides in a particular nucleic acid sequence asset forth herein (e.g. a particular polypeptide sequence characterizedby a SEQ. ID. NO. in the sequence listings), after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity. An alignment for purposes of determiningpercent nucleic acid sequence identity can be carried out according toprocedures known in the art, as described for example in EP 1 241 179B1. For example, a skilled person can use publicly available computersoftware, such as using publicly available computer software such asBLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. A skilled personcan determine appropriate parameters for measuring alignment, includingany algorithms needed to achieve maximal alignment over the full lengthof the sequences subjected to comparison. According to a preferredembodiment, the % identity values can be generated using the WU-BLAST-2computer program, as described for example in EP 1 241 179 B1. Accordingto a preferred embodiment, the following computer program and parametersare used: The identity values used herein are generated by the BLASTNmodule of WU-BLAST-2 set to the default parameters, with overlap spanand overlap fraction set to 1 and 0.125, respectively. A % nucleic acidsequence identity value can be obtained by dividing (a) the number ofmatching identical nucleotides between a particular nucleic acidsequence as set forth herein which is subjected to comparison (e.g. aparticular nucleic acid sequence characterized by a SEQ. ID. NO. in thesequence listings), and the comparison nucleic acid molecule of interestto be compared, for example the number of matching identical nucleotidesas determined by WU-BLAST-2, by (b) the total number of nucleotideresidues of the particular nucleic acid sequence as set forth hereinwhich is subjected to comparison (e.g. a particular nucleic acidsequence characterized by a SEQ. ID. NO. in the sequence listings).

In particular, sequence identity can be determined over the full lengthof a respective amino acid sequence as set forth in one of SEQ ID NO: 1to 49 or over the full length of a respective nucleotide sequence as setforth in one of SEQ ID NO: 50 to 128.

The term “positives”, in the context of sequence comparison performed asdescribed above and in EP 1 241 179 81, includes residues in thesequences compared that are not identical but have similar properties(e.g. as a result of conservative substitutions). The % value ofpositives is determined by the fraction of residues scoring a positivevalue in the BLOSUM 62 matrix divided by the total number of residues inthe aligned region.

Moreover, an antibody of the present invention specifically binds CD26and can have a heavy chain variable region, which heavy chain variableregion comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 22 to 47 and variant(s) thereof. A variant ofan amino acid sequence selected from the group consisting of SEQ ID NOs:22 to 47 can have at least 90%, preferably at least 98%, more preferablyat least 99% sequence identity to the amino acid sequence selected fromthe group consisting of SEQ ID NOs: 22 to 47. Alternatively, a variantof an amino acid sequence selected from the group consisting of SEQ IDNOs: 22 to 47 can be an amino acid sequence, wherein not more than 8,preferably not more than 5, more preferably 1 amino acid residue(s),respectively, have been deleted, inserted or replaced by an amino acidresidue different from the replaced amino acid residue. In oneembodiment, the amino acid replacement is a conservative change. Inparticular, an anti-CD26 antibody of the present invention can be anantibody, wherein the heavy chain variable region can comprise orconsist of an amino acid sequence selected from the group consisting ofSEQ ID NOs: 22 to 47 or variants thereof. Furthermore, an antibody ofthe present invention specifically binds CD26 and can have a heavy chainvariable region, which heavy chain variable region comprises amino acidresidues 7-112 of SEQ ID NO: 26. In one embodiment, an antibody of thepresent invention can be a chimeric antibody comprising human lightchain and heavy chain constant regions.

Furthermore, an antibody of the present invention can comprise a lightchain constant region, said light chain constant region comprising anamino acid sequence selected from the group consisting of SEQ ID NO: 48and variant(s) thereof. A variant of the amino acid sequence as setforth in SEQ ID NO: 48 can have at least 90%, preferably at least 98%,more preferably at least 99% sequence identity to SEQ ID NO: 48.Alternatively, a variant of SEQ ID NO: 48 can be an amino acid sequenceof SEQ ID NO: 48, wherein not more than 8, preferably not more than 5,more preferably 1 amino acid residue(s), respectively, have beendeleted, inserted or replaced by an amino acid residue different fromthe replaced amino acid residue. In one embodiment, the amino acidreplacement is a conservative change. In particular, an antibody of thepresent invention can be an antibody, wherein the light chain constantregion can comprise or consist of an amino acid sequence set forth inSEQ ID NO: 48 or a variant thereof.

In an alternative embodiment, an antibody of the present invention canbe a chimeric antibody comprising human light chain constant regions.

Moreover, an antibody of the present invention can comprise an aminoacid sequence set forth in SEQ ID NO: 49, or a variant thereof. Avariant of the amino acid sequence as set forth in SEQ ID NO: 49 mayhave at least 90%, preferably at least 98%, more preferably at least 99%sequence identity to SEQ ID NO: 49. Alternatively, a variant of SEQ IDNO: 49 can be an amino acid sequence, wherein not more than 8,preferably not more than 5, more preferably 1 amino acid residue(s),respectively, have been deleted, inserted or replaced by an amino acidresidue different from the replaced amino acid residue. In oneembodiment, the amino acid replacement is a conservative change. Inparticular, an antibody of the present invention can be an antibody,wherein the CH1-CH2-CH3 chain can comprise or consist of an amino acidsequence set forth in SEQ ID NO: 49 or a variant thereof.

In an alternative embodiment, an antibody of the present invention canbe a chimeric antibody comprising human heavy chain constant region.

In particular, an antibody of the present invention can be of IgG 2Bclass, and can comprise an amino acid sequence set forth in SEQ ID NO:49, or a variant thereof, as defined supra.

As may be seen from the sequence listings annexed, a group of sequenceshas been identified for heavy chain variable region (VH), and 2different groups of sequences have been identified for light chainvariable region (VL). Alignments of sequences of each group, showingtheir similarity, are shown in FIG. 3. A VL sequence (SEQ. ID. NO: 4)present in high frequency in antibodies of the present invention hasbeen identified and the corresponding CL sequence (SEQ. ID. NO: 48) hasbeen recognized. A VH sequence present in high frequency has beenidentified (SEQ. ID. NO: 26) and the corresponding CH1-CH2-CH3 sequence(SEQ. ID. NO: 49) has been recognized.

An antibody of the present invention can be an antibody whichspecifically binds to CD26, comprises a light chain variable regioncomprising an amino acid sequence set forth in SEQ ID NO: 4 or a variantthereof and or an amino acid sequence set forth in SEQ ID NO: 5 or avariant thereof and comprises a light chain constant region comprisingan amino acid sequence set forth in SEQ ID NO: 48 or a variant thereof.An antibody of the present invention can be an antibody whichspecifically binds CD26, comprises a heavy chain variable regioncomprising an amino acid sequence set forth in SEQ ID NO: 26 or avariant thereof and comprises a CH1-CH2-CH3 chain comprising an aminoacid sequence set forth in SEQ ID NO: 49 or a variant thereof.Furthermore, an antibody of the present invention can be an antibodywhich specifically binds CD26, comprises a light chain variable regioncomprising an amino acid sequence set forth in SEQ ID NO: 4 or a variantthereof and optionally further comprises a light chain constant regioncomprising an amino acid sequence set forth in SEQ ID NO: 48 or avariant thereof and optionally further comprises a CH1-CH2-CH3 chaincomprising an amino acid sequence set forth in SEQ ID NO: 49 or avariant thereof and optionally further comprises a heavy chain variableregion comprising an amino acid sequence set forth in SEQ ID NO: 26 or avariant thereof. In particular, an antibody of the present invention cancomprise a light chain variable region comprising an amino acid sequenceset forth in SEQ ID NO: 4 or a variant thereof, a light chain constantregion comprising an amino acid sequence set forth in SEQ ID NO: 48 or avariant thereof, a CH1-CH2-CH3 chain comprising an amino acid sequenceset forth in SEQ ID NO: 49 or a variant thereof and a heavy chainvariable region comprising an amino acid sequence set forth in SEQ IDNO: 26 or a variant thereof. Alternatively, an antibody of the presentinvention can be a chimeric antibody comprising a human light chainand/or heavy chain constant region.

In particular, an antibody of the present invention can be an antibodywhich specifically binds to CD26, comprises a light chain variableregion comprising amino acid residues 8-104 of SEQ ID NO: 4, a lightchain constant region comprising an amino acid sequence set forth in SEQID NO: 48 or a variant thereof, a CH1-CH2-CH3 chain comprising an aminoacid sequence set forth in SEQ ID NO: 49 or a variant thereof and aheavy chain variable region comprising amino acid residues 7-112 of SEQID NO: 26. Alternatively, an antibody of the present invention can be achimeric antibody comprising a human light chain and/or heavy chainconstant region.

In particular, an antibody of the present invention can be an antibodywhich specifically binds to CD26, comprises a light chain variableregion comprising an amino acid sequence set forth in SEQ ID NO: 5 or avariant thereof, a CH1-CH2-CH3 chain comprising an amino acid sequenceset forth in SEQ ID NO: 49 or a variant thereof and a heavy chainvariable region comprising amino acid residues 7-112 of SEQ ID NO: 22.Alternatively, an antibody of the present invention can be a chimericantibody comprising a human light chain and/or heavy chain constantregion.

An antibody of the present invention can be in particular an antibodywhich specifically binds to CD26, comprises a light chain variableregion comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21 or variant(s) thereof and comprises a light chainconstant region comprising an amino acid sequence set forth in SEQ IDNO: 48 or a variant thereof and comprises a CH1-CH2-CH3 chain comprisingan amino acid sequence set forth in SEQ ID NO: 49 or a variant thereof;optionally, this antibody furthermore comprises a heavy chain variableregion comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO: 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 or variant(s)thereof. Alternatively, an antibody of the present invention can be achimeric antibody comprising a human light chain and/or heavy chainconstant region.

An antibody of the present invention can be in particular an antibodywhich specifically binds to CD26. comprises a light chain variableregion comprising an amino acid sequence set forth in SEQ ID NO: 4 orvariant(s) thereof and comprises a light chain constant regioncomprising an amino acid sequence set forth in SEQ ID NO: 48 or avariant thereof and comprises a CH1-CH2-CH3 chain comprising an aminoacid sequence set forth in SEQ ID NO: 49 or a variant thereof;optionally, this antibody furthermore comprises a heavy chain variableregion comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO: 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 or variant(s)thereof. Alternatively, an antibody of the present invention can be achimeric antibody comprising a human light chain and/or heavy chainconstant region.

An antibody of the present invention can be in particular an antibodywhich specifically binds to CD26, comprises a light chain variableregion comprising an amino acid sequence set forth in SEQ ID NO: 5 orvariant(s) thereof and comprises a CH1-CH2-CH3 chain comprising an aminoacid sequence set forth in SEQ ID NO: 49 or a variant thereof; thisantibody can optionally furthermore comprise a heavy chain variableregion comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO: 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 or variant(s)thereof. Alternatively, an antibody of the present invention can be achimeric antibody comprising a human light chain and/or heavy chainconstant region.

An antibody of the present invention can be in particular an antibodywhich specifically binds to CD26, comprises a heavy chain variableregion comprising an amino acid sequence set forth in SEQ ID NO: 26 orvariant(s) thereof and comprises a light chain constant regioncomprising an amino acid sequence set forth in SEQ ID NO: 48 or avariant thereof and comprises a CH1-CH2-CH3 chain comprising an aminoacid sequence set forth in SEQ ID NO. 49 or a variant thereof; thisantibody can optionally furthermore comprise a light chain variableregion comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21 or variant(s) thereof. Alternatively, an antibody ofthe present invention can be a chimeric antibody comprising a humanlight chain and/or heavy chain constant region.

A variant of any one of the sequences selected from the group ofsequences SEQ ID NO: 1 to 128 can have at least 90%, preferably at least98%, more preferably at least 99% sequence identity to said sequenceselected from said group of sequences.

Moreover, an antibody of the present invention specifically binds CD26and can comprise a heavy chain variable region and a light chainvariable region, wherein the amino acid sequences of the heavy chainvariable region are those of a heavy chain variable region of anantibody produced by the hybridoma cell line deposited at CBA-ICLC ofGenoa (Italy) as PD 12002, and the amino acid sequences of the lightchain variable region are those of a light chain variable region of anor this antibody produced by the hybridoma cell line deposited as PD12002. In particular, the amino acid sequences of the heavy chain can bethose of a heavy chain of an antibody produced by the hybridoma cellline deposited as PD 12002, and/or the amino acid sequences of the lightchain can be those of a light chain of an or this antibody produced bythe hybridoma cell line deposited as PD 12002. In particular, theantibody of the present invention specifically binding to CD26 cancomprise the same heavy chain sequences and the same light chainsequences as an antibody produced by the hybridoma cell line depositedas PD 12002. Alternatively, an antibody of the present invention can bea chimeric antibody comprising a human light chain and/or heavy chainconstant region.

An anti-CD26 antibody of the present invention (e.g. an antibody thatspecifically binds CD26 and comprises a heavy chain variable regioncomprising SEQ ID NO: 1, and/or comprises a light chain variable regioncomprising SEQ ID NO: 2 and/or SEQ ID NO: 3) can be an antibody, whereinfurthermore the amino acid sequences of the heavy chain variable regioncan be those of a heavy chain variable region of an antibody produced bythe hybridoma cell line deposited at CBA-ICLC of Genoa (Italy) as PD12002, and the amino acid sequences of the light chain variable regioncan be those of a light chain variable region of an antibody produced bythe hybridoma cell line deposited as PD 12002. Optionally, the aminoacid sequence of the CDR3 of the heavy chain variable region can be thatof a heavy chain variable region of an antibody produced by thehybridoma cell line deposited as PD 12002. Optionally, the amino acidsequence of the CDR3 of the light chain variable region can be that of alight chain variable region of an antibody produced by the hybridomacell line deposited as PD 12002. Alternatively, an antibody of thepresent invention can be a chimeric antibody comprising a human lightchain and/or heavy chain constant region.

The term “hybridoma cell line” also includes the progeny of thehybridoma cell line, whether or not the progeny is identical inmorphology or in genetic make-up. Because certain modifications mayoccur, for example due to mutation and/or environmental influences, suchprogeny may not be identical to the parent cell line. Preferably, cellprogeny of this hybridoma cell line will produce an antibody, or anantibody fragment, capable of binding to CD26, especially to human CD26,in particular will produce an antibody of the present invention.Moreover, the term “hybridoma cell line” can also encompass mixtures ofhybridoma cell lines, producing an antibody mixture.

According to another aspect, the present invention provides an antibodymixture. The present invention also provides a composition, inparticular an isolated composition, comprising the antibody mixture.This antibody mixture can comprise at least two different antibodies,which at least two different antibodies preferably can specifically bindto CD26, especially human CD26. Optionally, at least one antibody whichdoes not bind to CD26 can be present in the antibody mixture. Accordingto one embodiment, at least two or all antibodies of the composition canspecifically bind to CD26, especially human CD26. In particular, one ormore or all of the antibodies present in the antibody mixture can beantibodies of the present invention and can optionally have furtherfeatures of the antibodies of the present invention as disclosed supra.This antibody mixture can comprise a first antibody, said first antibodycomprising a light chain variable region comprising the sequence setforth in SEQ ID NO: 2 and/or the sequence set forth in SEQ ID NO: 3, inparticular in SEQ ID NO: 3, and a second antibody, said second antibodycomprising a light chain variable region comprising the sequence setforth in SEQ ID NO: 2. The first antibody and/or the second antibody canbe antibodies of the present invention and can optionally have one ormore further features of antibodies of present invention as discussed indetail supra. In one embodiment, the antibody mixture comprises orconsists of antibodies having a heavy chain variable region comprising aVH CDR1 of SEQ ID NO: 133, a VH CDR2 of SEQ ID NO: 134, and a VH CDR3 ofSEQ ID NO: 1.

According to a preferred embodiment of the invention, the antibodymixture comprises at least one or comprises or consists of twoantibodies having the following combination of sequences (each antibodycomprising one of the light chain sequences indicated below togetherwith the heavy chain sequence indicated below):

One of the following two different light chains comprising

a) Light chain variable region comprising SEQ ID NO: 4, or a variant ofSEQ ID NO: 4, in particular any of SEQ ID NO: 6 to SEQ ID NO: 21,together with light chain constant region comprising SEQ ID NO: 48; or

b) Light chain variable region comprising SEQ ID NO: 5,

Together with a heavy chain comprising:

Heavy chain variable region comprising SEQ ID NO: 26 or a variant of SEQID NO: 26, in particular any sequence selected from of SEQ ID NO: 22 toSEQ ID NO: 25 and SEQ ID NO: 27 to SEQ ID NO: 47, together with heavychain constant region (CH1-CH2-CH3) comprising SEQ ID NO: 49.

In one embodiment, the antibody mixture comprises or consists of theantibodies produced by the PD 12002 hybridoma deposit. In oneembodiment, the isolated composition comprises at least one of theantibodies produced by the PD 12002 hybridoma deposit, in particularcomprises the antibodies produced by the PD 12002 hybridoma deposit.

During the numerous experiments performed by the present inventors, theantibodies produced by the PD 12002 hybridoma deposit showed up to havesurprisingly beneficial activities for use as a medicament, inparticular for promoting engraftment after haematopoietic stem celltransplantation and/or preventing and/or treating at least one ofGraft-versus-Host Disease and Aplastic Anemia.

The PD 12002 hybridoma cell line as deposited is stable in storage andculture and has been cultivated and verified for stability and identityover more than 5 years.

In another aspect, the invention provides an agent (in particularantibody or fragment thereof) that competes for specific binding toCD26, in particular to human CD26, with an antibody in a competitivebinding assay (e.g., in an in vitro competitive binding assay), whereinthe antibody is an antibody of the present invention; in particular,this agent can compete for specific binding to CD26, in particular tohuman CD26, with an antibody which can comprise

a) a heavy chain variable region comprising the sequence set forth inSEQ ID NO: 1 and/or a light chain variable region comprising thesequence set forth in SEQ ID NO: 2 and/or the sequence set forth in SEQID NO: 3;

b) a heavy chain variable region of an antibody produced by thehybridoma cell line deposited at CBA-ICLC, Genoa, Italy as PD12002 and alight chain variable region of said antibody produced by said hybridomacell line deposited at CBA-ICLC, Genoa, Italy as PD12002. According toone embodiment, the antibody can be an antibody which can comprisea) a heavy chain variable region comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 22 to 47 andvariant(s) thereof, and/or a light chain variable region comprising anamino acid sequence selected from the group consisting of SEQ ID NOs: 4,5, 6 to 21, and variant(s) thereof, or an antibody which can compriseb) a heavy chain and a light chain of an antibody produced by thehybridoma cell line deposited at CBA-ICLC, Genoa, Italy as PD12002.According to a preferred embodiment, the invention provides an agent (inparticular antibody or fragment thereof) that competes for specificbinding to CD26, in particular to human CD26, in a competitive bindingassay (e.g., in an in vitro competitive binding assay) with an antibodycomprising a heavy chain variable region sequence ID NO: 26 and a lightchain variable region sequence ID NO: 4 and/or sequence ID NO: 5.Competitive binding assay can be used to determine whether twoantibodies bind the same epitope by recognizing identical or stericallyoverlapping epitopes (Dong et. al 1998). Any competitive binding assayknown to one of skill can be used to identify an agent that competes forspecific binding to CD26 with an antibody of the present invention. Forexample, assays in which a CD26 antigen is immobilized on a multi-wellplate and the ability of unlabelled antibody to block the binding oflabelled antibodies is measured can be used. Common labels for suchcompetition assays are radioactive labels or enzyme labels.

According to another aspect, an isolated nucleic acid moleculecomprising (a) a nucleotide sequence encoding an antibody of the presentinvention or (b) a nucleotide sequence complementary to (a) is providedby the inventors.

In the context of the present invention, the term “nucleic acidmolecule” is used as known in the art and can in particular refer to twoor more nucleotides or nucleotide analogs linked by a covalent bond. Theterm “nucleic acid molecule” encompasses oligonucleotides, whichgenerally comprise not more than about fifty nucleotides, andpolynucleotides, which can have essentially any length. Furthermore, theterm “nucleic acid molecule” can encompass DNA, such as a cDNA or agene, or RNA. The nucleotides comprising a nucleic acid molecule can befor example selected from the group comprising naturally occurringdeoxyribonucleotides, ribonucleotides and nucleotide analogs, such asnon-naturally occurring synthetic nucleotides or modified naturallyoccurring nucleotides. Nucleotide analogs are known in the art and aredescribed e.g. in Lin et al., 1994, Nucl. Acids Res. 22:5220-5234;Jellinek et al., 1995, Biochem. 34:11363-11372; Pagratis et al., 1997,Nature Biotechnol. 15:68-73.

An “isolated” compound or composition, such as e.g. a polypeptide,antibody, nucleic acid molecule, vector, cell, or a mixture thereof, canbe in particular a compound or composition which is present in a formnot found in nature. Isolated compounds (e.g. polypeptides, nucleic acidmolecules, antibodies, vectors, cells) or compositions include thosewhich have been purified to an extent that they are no longer in a formin which they are found in nature.

The present invention also provides an isolated nucleic acid moleculecomprising a nucleotide sequence selected from the group consisting ofSEQ ID NOs: 50 to 128 and variant(s) thereof. A variant of thenucleotide sequence selected from the group consisting of SEQ ID NOs: 50to 128 can have at least 90%, preferably at least 98%, more preferablyat least 99% sequence identity to the nucleotide sequence selected fromthe group consisting of SEQ ID NOs: 50 to 128. A nucleotide sequenceselected from the group consisting of SEQ ID NOs: 50 to 77 or a variantthereof can encode a light chain variable region (VI) or a sectionthereof. Alternatively, a variant of a nucleotide sequence selected fromthe group consisting of SEQ ID NOs: 50 to 128 can be a nucleotidesequence selected from the group consisting of SEQ ID NOs: 50 to 128,wherein not more than 12, preferably not more than 5, more preferablynot more than 1 nucleotide residue(s), respectively, have been deleted,inserted or replaced by a nucleotide residue different from the replacednucleotide residue. A nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 78 to 126 or a variant thereof can encode aheavy chain variable region (VH) or a section thereof. The nucleotidesequence set forth in SEQ ID NO: 127 or a variant thereof can encode alight chain constant region (CL) or a section thereof. The nucleotidesequence set forth in SEQ ID NO. 128 or a variant thereof can encode aCH1-CH2-CH3 chain or a section thereof. According to an embodiment, thenucleotide sequence encoding the VL chain can be a productive IGK (Igkappa locus) rearranged sequence (in-frame junction and no stop codon).According to an embodiment, the nucleotide sequence encoding the VHchain can be a productive IGH (Ig heavy chain gene) rearranged sequence(in-frame junction and no stop codon).

The before-mentioned variants of the nucleic acid molecules can be forexample obtained by means of “parsimonious mutagenesis” (Shier, R., etal., 1996, Gene 169: 147) or by means of other methods of random ordirected mutagenesis of nucleotide sequences of the present invention(Marks, J. D., et al., 1992, J. Biol. Chem. 267: 16007) performed inorder to improve some of the properties of antibodies, as for instancethe affinity, while preferably maintaining binding specificity for CD26.

The nucleic acid molecules of the present invention can be cloned invectors suitable for their amplification, further mutagenesis ormodification or expression. The present invention also provides a vectorcomprising a nucleotide sequence encoding for an antibody of the presentinvention. Preferably, the vector is capable of effectively expressingan antibody according to the present invention. In particular, a nucleicacid vector can comprise a first nucleic acid molecule covalently andoperatively linked to a second nucleic acid molecule such that a hostcontaining the vector expresses the polypeptide coded for by the firstnucleic acid molecule, the first nucleic acid molecule being a nucleicacid molecule according to the present invention, in particular anucleic acid molecule comprising a nucleotide sequence selected from thegroup consisting of SEQ ID NOs: 50 to 128 and variant(s) thereof. Thesevectors can be used for the preparation of recombinant antibodies or ofchimeric proteins in a suitable host and following methods known in theart.

In accordance to a currently preferred embodiment of the presentinvention, the recombinant antibodies are preferably cloned andexpressed in prokaryotic cells or eukaryotic host cells: particularlypreferred is E. coli, but also other prokaryotic cells can be used, suchas B. subtilis, P. pastoris, K lactis, or eukaryotic cells of plant oranimal origin, in particular of murine origin.

According to yet another aspect, an expression vector comprising anucleic acid molecule of the present invention, wherein said nucleicacid molecule is operatively linked to an expression control sequence,is provided.

The term “vector” can in particular refer to a molecule, for example anucleic acid molecule, plasmid, or virus, used to transfer codinginformation to a host cell. In particular, a “vector” can be a nucleicacid molecule, preferably self-replicating, which can transfer aninserted nucleic acid molecule into and/or between host cells. Examplesof vectors include, but are not limited to, viral vector, whereinadditional DNA segment(s) can be ligated into the viral genome, nakedDNA or RNA expression vectors, DNA or RNA expression vectorsencapsulated in liposomes, plasmid, such as for example a circulardouble stranded DNA loop into which additional DNA segment(s) can beligated, cosmid or phage vector, a vector, which is capable ofautonomous replication in a host cell into which the vector has beenintroduced, and DNA or RNA expression vectors associated with cationiccondensing agents. In particular, a vector can be integrated into thegenome of a host cell upon introduction into the host cell, what permitsthat it is subsequently replicated along with the host genome. Inparticular, the term “vector” encompasses expression vectors. As usedherein, the term “expression vector” can in particular refer to a vectorwhich is capable of directing the expression of one or more gene(s) towhich the expression vector is operatively inked. Expression vectorscontaining nucleotide sequences as described herein can be optimised forexpression in a host cell, in particular by insertion of suitableregulator regions, promoters, transcriptional terminators or activators,or replication origin.

In the context of the present application, components which are“operatively linked” can be in particular components which are in arelationship permitting the components to function in their intendedmanner. An expression control sequence operatively linked to a codingsequence can be in particular ligated such that expression of the codingsequence is achieved under conditions compatible with the one or moreexpression control sequence(s). The term “expression control sequence”encompasses, but is not limited to one or more nucleotide sequence(s)that regulate the expression of a nucleotide sequence to which theexpression control sequence is operatively linked. Operatively linkedexpression control sequences can include, but are not limited to,expression control sequences that are contiguous with the gene ofinterest and expression control sequences that act in trans or at adistance for controlling a gene of interest.

According to one embodiment, an expression control sequence operativelylinked to a nucleic acid sequence can control and regulate thetranscription and, when appropriate, translation of the nucleic acidsequence. Expression control sequences can include, but are not limitedto, one or more sequences selected from the group consisting of promotersequences, enhancer sequences, transcription terminators, splicingsignal for intron(s), if intron(s) are present, start codon, inparticular in front of a protein-encoding gene, sequences ensuring aproper translation of mRNA. and stop codons. According to oneembodiment, an expression vector can for example contain an origin ofreplication, a promoter, and optionally one or more genes which allowphenotypic selection of transformed cells.

According to yet another aspect, the present invention provides arecombinant host cell comprising a nucleic acid molecule of the presentinvention.

In the context of the present application, the term “host cell” can inparticular refer to a cell that has been transformed, or is capable ofbeing transformed, with a nucleic acid sequence. After having beentransformed a host cell can express a selected gene of interest. Theterm “host cell” not only encompasses the cell obtained aftertransformation, but also includes the progeny of the cell obtained aftertransformation, whether or not the progeny is identical in morphology orin genetic make-up to the original parent cell. Because certainmodifications may occur, for example due to mutation and/orenvironmental influences, such progeny may not be identical to theparent cell. Preferably, the progeny produces an antibody or an antibodyfragment, which are capable of binding to CD26, especially human CD26.The term “host cell” can also encompass mixtures of host cells. Inmixtures of host cells, the host cells may produce one antibody or onefragment thereof or two or more different antibodies or fragmentsthereof.

An antibody of the present invention can be an antibody produced fromthe hybridoma cell line deposited at CBA-ICLC of Genoa (Italy) as PD12002 or a derivative of said hybridoma cell line. In particular, aderivative of the hybridoma cell line deposited as PD 12002 is a cellline comprising a polynucleotide comprising a sequence, which is avariant of one or more of SEQ ID NOs: 50 to 128. An antibody of thepresent invention can be an antibody that binds the epitope bound by anantibody produced by the hybridoma cell line deposited at CBA-ICLC ofGenoa (Italy) as PD 12002. As used herein, the term “epitope” may inparticular refer to a portion of an antigen capable of being recognizedand specifically bound by a particular antibody. Usually, an epitope caninclude at least 3, and more usually, at least 4, or 8 to 10 amino acidsin a particular spatial conformation. Since an antibody can recognize anantigenic peptide or polypeptide in its tertiary form, the amino acidscomprising an epitope need not be contiguous, and in some cases, may noteven be on the same peptide chain. In the present invention, a peptideor polypeptide epitope recognized by antibodies of the present inventioncontains a sequence of at least 4, at least 5, at least 6, at least 7,at least 8, at least 9, at least 10, at least 15, at least 20, at least25, or between about 5 to about 30, about 10 to about 30 or about 15 toabout 35 contiguous or non-contiguous amino acids of CD26.

In one embodiment, an anti-CD26 monoclonal antibody of the presentinvention is capable of binding a CD26 epitope comprising one or moreamino acid sequences selected from the group consisting of SEQ ID NO:146-159. In specific embodiments the epitope can be a continuous epitopeor a discontinuous epitope. In one embodiment, an anti-CD26 monoclonalantibody of the present invention is capable of binding a CD26 epitopecomprising one or more amino acid sequences selected from the groupconsisting SEQ ID NO: 146-148, 152, 154, 158, and 159. In oneembodiment, an anti-CD26 monoclonal antibody of the present invention iscapable of binding a CD26 epitope comprising an amino acid sequencesselected from the group consisting SEQ ID NO: 146-148, 152, 154, 158,and 159. In another embodiment, an anti-CD26 monoclonal antibody of thepresent invention is capable of binding a CD26 epitope comprising SEQ IDNO: 146. In a further embodiment, an anti-CD26 monoclonal antibody ofthe present invention is capable of binding a CD26 epitope comprisingSEQ ID NO: 146 and or more amino acid sequences selected from the groupconsisting of SEQ ID NO: 147, 148, 152, 154, and 159. In a particularembodiment, an anti-CD26 monoclonal antibody of the present invention iscapable of binding a CD26 epitope comprising SEQ ID NOs: 146, 147, and148; or SEQ ID NOs 146, 147, and 152; or SEQ ID NOs 146 and 147; or SEQID NOs 146 and 152.

According to yet another aspect, the present inventors provide anantibody produced from the hybridoma cell line deposited at CBA-ICLC ofGenoa (Italy) as PD 12002 or a derivative of said hybridoma cell line.

According to another aspect, an antibody that binds the epitope bound byan antibody produced by the hybridoma cell line deposited at CBA-ICLC ofGenoa (Italy) as PD 12002 is provided.

According to yet another aspect, a process of manufacturing an antibodyof the present invention is provided. This process comprises the stepsof:

-   (i) providing a host cell of the present invention, in particular    the hybridoma cell line deposited at CBA-ICLC of Genoa (Italy) as PD    12002;-   (ii) culturing the host cell of the present invention in a culture    medium; and-   (iii) obtaining the antibody from the medium;-   (iv) optionally purifying the antibody, for example by filtration    and/or nanofiltration.

According to one embodiment, transgenic animals that have beengenetically engineered to produce antibodies, in particular humanantibodies, can be used to generate an antibody against an immunogenictarget as mentioned in the present application, in particular anantibody to CD26. Transgenic animals and antibodies produced by thesetransgenic animals are obtainable using techniques known in the art, inparticular using standard immunization protocols, as described e.g. inUS 2010/0196266 A1. Methods for obtaining human antibodies fromtransgenic animals, in particular transgenic mice, are described e.g. inGreen et al., Nature Genet. 7:13 (1994), Lonberg et al., Nature 368:856(1994), and Taylor et al., Int. Immun. 6:579 (1994). A non-limitingexample for a transgenic animal producing antibodies, in particularhuman antibodies, is a transgenic mouse, in particular the XenoMouse®from Abgenix (Fremont, Calif., USA), as described for example in Greenet al., 1999. J. Immunol. Methods 231:11-23). In a transgenic animal,such as the XenoMouse®, the antibody genes of a non-human mammalsubjected to genetically engineering, for example the mouse antibodygenes, have been inactivated and replaced by functional human antibodygenes, while the remainder of immune system of the non-human mammalsubjected to genetic engineering, for example the mouse immune system,remains intact.

Human antibodies produced by transgenic animals can show therapeuticpotential, while retaining the pharmacokinetic properties of normalhuman antibodies (Green et al., 1999, supra, US 2010/0196266 A1). Theuse of the XenoMouse® system has been merely exemplarily mentioned inthe present application for producing antibodies. On the basis of thegeneral knowledge in the art, a skilled person can also use anothertransgenic animal, in particular e.g. transgenic rodents, sheep, goatsor cows, for producing antibodies of the present invention, inparticular human antibodies.

Unless explicitly indicated otherwise, all before-mentioned techniquesare exemplary techniques and any known method for producing antibodiesor antibody fragments can be utilized. For carrying out the presentinvention, unless indicated otherwise, conventional techniques of cellbiology, organic chemistry, biochemistry, molecular biology, cellculture, microbiology, protein chemistry, recombinant DNA, andimmunology, can be employed. Such conventional techniques are forexample described in: Molecular Cloning: A Laboratory Manual, 2^(nd)edition (Sambrook et al., Eds.), 1989; Oligonucleotide Synthesis, (M. J.Gait, Ed.), 1984; U.S. Pat. No. 4,683,195 (Mullis et al.); Nucleic AcidHybridization, (B. D. Hames et al.), 1984; Methods in Enzymology,Volumes 154 and 155 (Wu et al.), Academic Press, New York; Transcriptionand Translation, (B. D. Hames and S. J. Higgins), 1984; Culture ofAnimal Cells (R. I. Freshney, ed.), 1987; Immobilized Cells and Enzymes,IRL Press, 1986; A Practical Guide to Molecular Cloning (B. Perbal),1984; Gene Transfer Vectors for Mammalian Cells (J. H. Miller and M. P.Calos, Eds.), 1987; Immunochemical Methods in Cell and Molecular Biology(Mayer and Walker, eds.), 1987; Handbook of Experiment Immunology,Volumes I-IV (D. M. Weir and C. C. Blackwell, eds.), 1986; Manipulatingthe Mouse Embryo, 1986.

Unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular.

According to another aspect, antibodies of the present invention areantibodies for use as a medicament. In particular, antibodies of thepresent invention are antibodies for use in preventing and/or treatingGraft-versus-Host Disease (GvHD), preferably after haematopoietic stemcell transplantation. Furthermore, the antibodies of the presentinvention are antibodies for use in preventing and/or treating AplasticAnemia, preferably Severe Aplastic Anemia. Moreover, an antibody of thepresent invention can be an antibody for use in promoting engraftmentafter haematopoietic stem cell transplantation. An antibody of thepresent invention can also be an antibody which is for use in preventingand/or treating Graft-versus-Host Disease (GvHD), preferably afterhaematopoietic stem cell transplantation, and for use in preventingand/or treating Aplastic Anemia, preferably Severe Aplastic Anemia, andfor use in promoting engraftment after haematopoietic stem celltransplantation.

Graft-versus-Host Disease, Aplastic Anemia and the condition of asubject before and/or during and/or after haematopoietic stem celltransplantation are considered as being CD26 mediated disease(s),disorder(s) or condition(s), i.e. disease(s), disorder(s) orcondition(s) which can be treated and/or prevented by an administrationof agent(s), in particular one or more antibodies that can specificallybind to CD26, especially human CD26.

Moreover, the present invention also provides an antibody mixture of thepresent invention, in particular a composition, especially an isolatedcomposition, comprising an antibody mixture of the present invention,for use as a medicament. In particular, the antibody mixture, especiallythe isolated composition comprising an antibody mixture of theinvention, can be for use in promoting engraftment after haematopoieticstem cell transplantation, and/or for use in preventing and/or treatingGraft-versus-Host disease (GvHD), preferably after haematopoietic stemcell transplantation, and/or for use in preventing and/or treatingAplastic Anemia, preferably Severe Aplastic Anemia. In particular, theantibody mixture can comprise a first antibody of the present invention,said first antibody comprising a light chain variable region comprisingthe sequence set forth in SEQ ID NO: 2 and/or the sequence set forth inSEQ ID NO: 3, in particular the sequence set forth in SEQ ID NO: 3, anda second antibody of the present invention, said second antibodycomprising a light chain variable region comprising the sequence setforth in SEQ ID NO: 2. In one embodiment, the antibody mixture comprisesor consists of the antibodies produced by the PD 12002 hybridomadeposit. Furthermore, the antibody mixture can comprise an antibody(e.g., human or humanized antibody) of the present invention comprisingall 6 CDRs sequences of an antibody produced by the hybridoma cell linedeposited as PD12002.

Acquired aplastic anemia (AA) is a rare bone marrow failure statecharacterized by marrow hypocellularity and low peripheral blood cellcounts [Young N. S. et al., 1997 N Eng J Med 336:1365-1372]. Similar toother autoimmune diseases, antigen-specific T cells could be expandedfrom the bone marrow of AA patients and are likely to mediateorgan-specific cytotoxicity to haematopoietic stem cells and progenitorcells [Nakao S. et al., Blood 1997, 89:3691-3699].

Differentially expressed genes, which were exclusively found inBM-infiltrating T-cells, were classified into several functionalcategories. These differentially expressed genes included moleculesinvolved in immune responses as PF-4, CD26, Ncf-1, CCR2 and otherchemokine receptors and ligands. Moreover, it has been supposed that AAresults from auto aggressive destruction of haematopoietic stem cellsand progenitors mediated by T-cells recognizing inciting target antigens[Young N. S. et al., 1997, supra]. Several groups have identified clonalT-cell expansion [Zeng W. et al., Blood 1999, 93(9):3008-3016; Zeng W.et al., J Clin Invest 2001, 108(5):765-773; Risitano A. M. et al., Blood2002, 100(1):178-183], proinflammatory cytokine production [MaciejewskiJ. P. et al., Blood 1995, 85:3183-3190] and T-cell mediated cytotoxicityto CD34+ stem cells [Nakao S. et al., 1997, supra Maciejewski J. P.,Selleri C., Sato T. et al., Br J Haematol 1995, 91:245-252] supportingan antigen-driven T-cell response. The regulation of 483 genes alsodemonstrates that the bone marrow failure results from a rather complexgenetic program involving chemokines, cytokines, growth factors, andtheir receptors. Franzke and colleagues identify the induction ofseveral molecules playing key roles in the regulation of Th1 immuneresponses [Anke Franzke at al., BMC Genomics 2006, 7:263], such as CCR2and CX3CR1 [Charo I. F. at al., Microcirculation 2003, 10(3-4):259-264;Fraticelli P. at al., J Clin Invest 2001, 107(9):1173-1181], which arealso important in other autoimmune diseases such as multiple sclerosis[Lock C. et al., Nat Med 2002, 8(5):500-508; Jee Y. at al., JNeuroimmunol 2002, 128(1-2):49-57], and CD26, a surface-boundectopeptidase expressed at high levels on Th1 differentiated T-cells[Dang N. H. et al., Histol Histopathol 2002, 17(4):1213-1226; WillheimM. et al., J Allergy Clin Immunol 1997, 100:348-255].

While not wishing to be bound to any theory, it is currently assumedthat an antibody of the present invention capable of binding to CD26, inparticular capable of specifically binding to CD26, especially to humanCD26, might play a key role in the immunpathogenesis of AA and recoveryof haematopoiesis after immunosuppression.

A monoclonal antibody against CD26 antigen can be therefore used fortreating aplastic anemia (in particular congenital or acquired aplasticanemia), especially severe aplastic anemia.

The terms “treatment” and “prevention” as used in the presentapplication can in particular refer to any type of treatment orprevention that imparts a benefit to a subject afflicted with a disease,a disorder or a condition or at risk of developing a disease, a disorderor a condition, in particular at least one of Graft-versus-Host disease(GvHD) and Aplastic Anemia. Moreover, the terms “treatment” and“prevention” can also refer to any type of treatment or prevention thatimparts a benefit to a person with respect to engraftment afterhaematopoietic stem cell transplantation. The benefit imparted by thetreatment or the prevention can be the benefit of providing animprovement in the condition of the subject (for example in one or moresymptoms), the benefit of providing a delay in the progression of thedisease, disorder or condition to be treated and/or prevented, thebenefit of delaying the onset of one or more symptoms, the benefit ofalleviating the disease, disorder or condition to be treated and/orprevented and/or the benefit of providing a slower progression ofsymptoms, etc. Moreover, the terms “treatment” and “prevention” as usedin the present application are not necessarily meant to imply cure orcomplete abolition of symptoms.

The term “Graft-versus-Host disease” encompasses acute and/or chronicGraft-versus-Host disease, in particular Graft-versus-Host disease afterhaematopoietic stem cell transplantation. The term “Aplastic Anemia”encompasses both acquired and congenital aplastic anemia, as well assevere aplastic anemia.

The use of an antibody of the present invention can provide treatmentand/or prevention for human subjects, in particular for medicalpurposes, and for animal subjects, in particular for veterinary and drugscreening and development purposes. Suitable animal subjects includemammals, such as for example rabbits, primates, bovines, etc. Humansubjects are the most preferred. Human subjects include neonatal,infant, juvenile and adult subjects. The terms “patient” and “subject”are used interchangeably herein. As used herein, the term “patient” mayrefer to humans, but is not restricted to humans.

According to another aspect, the present invention provides apharmaceutical composition comprising at least one antibody of thepresent invention (for example one antibody, or two or more, or three ormore antibodies of the present invention) or an antibody mixturecomprising at least one antibody of the present invention or an isolatedcomposition comprising at least one antibody of the present invention,and optionally at least one pharmaceutically acceptable excipient. Inone embodiment, a pharmaceutical composition can comprise an antibody(e.g., human or humanized antibody) comprising the 6 CDRs sequences ofan antibody produced by the hybridoma cell line deposited as PD 12002.In another embodiment, a pharmaceutical composition can comprise anantibody (e.g., chimeric antibody) comprising the VH and VL regions ofan antibody produced by the hybridoma cell line deposited as PD 12002.In a further embodiment, a pharmaceutical composition can comprise anantibody mixture comprising a first antibody of the present invention,said first antibody comprising a light chain variable region comprisingthe sequence set forth in SEQ ID NO: 2 and/or the sequence set forth inSEQ ID NO: 3, in particular the sequence set forth in SEQ ID NO: 3, anda second antibody of the present invention, said second antibodycomprising a light chain variable region comprising the sequence setforth in SEQ ID NO: 2. The first and the second antibody can bedifferent antibodies, in particular the second antibody can have anamino acid sequence, wherein at least one amino acid residue has beendeleted, inserted or replaced by a different amino acid residue, whencompared to the amino acid sequence of the first antibody. Thepharmaceutical composition can comprise the at least one antibody of thepresent invention, for example an antibody of the present invention oran antibody mixture, or an isolated composition of the presentinvention, as an effective ingredient. In particular, the pharmaceuticalcomposition can comprise the at least one antibody of the presentinvention in an amount effective for treating and/or preventing at leastone of Graft-versus-Host disease, preferably after haematopoietic stemcell transplantation, and Aplastic Anemia, preferably Severe AplasticAnemia. Moreover, the pharmaceutical composition can comprise the atleast one antibody of the present invention in an amount effective forpromoting engraftment after haematopoietic stem cell transplantation.

The pharmaceutical composition comprising at least one antibody of thepresent invention is a pharmaceutical composition for use as amedicament. In particular, a pharmaceutical composition comprising atleast one antibody of the present invention is a pharmaceuticalcomposition for use in promoting engraftment after haematopoietic stemcell transplantation, and/or for use in preventing and/or treatingGraft-versus-Host disease (GvHD), preferably after haematopoietic stemcell transplantation, and/or for use in treating and/or preventingAplastic Anemia, preferably Severe Aplastic Anemia.

The pharmaceutical composition of the present invention can optionallycomprise one or more excipients, preferably pharmaceutically acceptableexcipients, in particular one or more diluents, preferablypharmaceutically acceptable diluents. Appropriate excipient(s), inparticular pharmaceutically acceptable excipient(s), can be chosen by askilled person on the basis of the general knowledge in the art and onthe basis of the teachings provided in the present application. Thediluent can be for example a pharmaceutically acceptable solvent or apharmaceutically acceptable solvent mixture, such as for example water.Examples of suitable excipients, in particular diluents, are well knownin the art and can be selected e.g. from the group comprising fluidscomprising a pharmaceutically acceptable buffering system, in particularsolutions comprising a pharmaceutically acceptable buffering system, forexample phosphate buffered saline solutions, water, saline, inparticular physiological saline, emulsions, such as oil/water emulsions,one or more wetting agents, sterile solutions, etc. A pharmaceuticalcomposition can also contain one or more pharmaceutically acceptablecarriers.

According to one embodiment, a pharmaceutical composition of the presentinvention can comprise at least one antibody of the present invention,such as at least one e.g. monoclonal or e.g. murine or e.g. monoclonaland murine anti-CD26 antibody according to the present invention, andwater and a phosphate buffer, preferably a buffer comprising M′H₂PO₄ andM″M′″HPO₄, wherein M′, M″ and M′″ can be independently selected from thegroup consisting of Na and K. This pharmaceutical composition canoptionally comprise one or more of NaCl, KCl and mixtures comprisingNaCl and KCl. The at least one antibody of the present invention cancomprise or consist of the antibodies produced by the PD 12002 hybridomadeposit or at least one antibody thereof. The pharmaceutical compositionof the present invention can be for intravenous administration, inparticular intravenous injection or intravenous infusion. In aparticular embodiment, a pharmaceutical composition of the presentinvention can comprise or consist of the antibodies produced by the PD12002 hybridoma deposit and DPBS.

According to one embodiment, a pharmaceutical composition of the presentinvention can comprise at least one antibody of the present invention,e.g. a monoclonal antibody anti CD26 according to the present invention,in particular the antibodies produced by the PD 12002 hybridoma depositor at least one antibody thereof. In one embodiment, the pharmaceuticalcomposition can comprise at least one antibody of the present invention,e.g. the antibodies produced by the PD 12002 hybridoma deposit or atleast one antibody thereof, one or more corticosteroids, one or moreantihistamines and saline, e.g. physiological saline (especially salinecontaining about 0.9% w/v NaCl). In particular, these aqueouspharmaceutical compositions can be administered by infusion, especiallyslow infusion, in particular by intravenous administration.

In one embodiment, a pharmaceutical composition of the inventioncomprises an antibody of the invention in a concentration range from 1mg/ml to 10 mg/ml, 1 mg/ml to 50 mg/ml, 1 mg/ml to 100 mg/ml, 10 mg/mlto 100 mg/ml, or 50 mg/ml to 100 mg/ml. In particular embodiment, apharmaceutical composition of the invention can comprise at least about1 mg/ml, at least about 1 mg/ml, at least about 5 mg/ml, at least about10 mg/ml, at least about 20 mg/ml, at least about 30 mg/ml, at leastabout 40 mg/ml, at least about 50 mg/ml, or at least about 100 mg/ml ofan antibody of the invention.

In one embodiment, pharmaceutical compositions can comprise the at leastone antibody of the present invention in amount between 1 mg of said atleast one antibody/m² body surface area per day and 4.5 mg of said atleast one antibody/m² body surface area per day for use in the treatmentand/or prevention of Graft-versus-Host disease or can comprise at leastone antibody of the present invention in an amount between 1 mg of saidat least one antibody/m² body surface area per day and 2 mg of said atleast one antibody/m² body surface area per day for use in the treatmentand/or prevention of Aplastic Anemia. Moreover, according to oneembodiment the pharmaceutical compositions can comprise currentlypreferred ranges of amounts and amounts of at least one antibody of thepresent invention as discussed herein.

The term “pharmaceutically acceptable” as used herein can in particularindicate that the “pharmaceutically acceptable” compound or“pharmaceutically acceptable” composition is suitable for administrationto a subject to achieve a treatment and/or prevention of a disease, of adisorder or of a condition, in particular of at least one ofGraft-versus-Host disease and Aplastic Anemia or to achieve promoting ofengraftment after haematopoietic stem cell transplantation, withoutunduly deleterious side effects in light of the severity of the diseaseand necessity of the treatment.

The term “effective amount” as used herein can in particular indicate anamount of the at least one antibody of the present invention, forexample of an antibody or of an antibody mixture, sufficient to producea desirable effect upon a patient inflicted with a disease, disorder ora condition, in particular at least one of Graft-versus-Host disease andAplastic Anemia, or to produce a desirable effect upon a patient withrespect to engraftment after haematopoietic stem cell transplantation,including improvement in the condition of the patient (e.g., in one ormore symptoms), delay in the progression of the disease, etc. Aneffective amount of an antibody described herein can in particular be anamount sufficient to ameliorate, reverse, stabilize, slow and/or delayprogression of Graft-versus-Host disease and/or Aplastic Anemia. Asknown in the art, an effective amount of, for example, an antibodyaccording to the present invention can vary, depending on, inter alia,patient history, administration for prevention or treatment purposes,target indication (Graft-versus-Host disease, Aplastic Anemia, etc.), aswell as other factors, such as the type (and/or dosage) of antibody.

A pharmaceutical composition of the present invention can be in solid orliquid form and can be, inter alia, in a form of one or more powder(s),one or more tablet(s), one or more fluids, in particular one or moresolution(s), or one or more aerosol(s). A pharmaceutical composition ofthe invention can also comprise one or more further biologically activeagent(s), such as for example active agent(s) for use in the treatmentand/or prevention of at least one of Graft-versus-Host disease andAplastic Anemia or active agent(s) for promoting engraftment afterhaematopoietic stem cell transplantation. The administration of apharmaceutical composition of the present invention can be for examplean administration selected from the group consisting of intraperitoneal,intravenous, parenteral, intrarenal, subcutaneous, topical,intrabronchial, intrapulmonary and intranasal administration and, ifdesired for local treatment, intralesional administration. A parenteraladministration can be for example an intraperitoneal, intradermal,intramuscular, subcutaneous, intravenous or intraarterial,administration. The compositions of the invention can also beadministered directly to the target site, e.g., by biolistic delivery tothe target site, like a specific organ afflicted with a disease,disorder or condition, in particular Graft-versus-Host disease.

In particular, said administration can be carried out by injectionand/or infusion and/or delivery. such as e.g. intravenous orintraperitoneal injection or infusion. The pharmaceutical compositioncan be present in the form of an injectable dosage form or a dosage formfor administration by infusion, in particular in the form of aninjectable dosage form for intravenous or intraperitoneal injection oran infusion dosage form for intravenous or intraperitonealadministration.

A pharmaceutical composition, in particular a pharmaceutical compositionin the form of an injectable dosage form, can comprise one or morepharmaceutically acceptable solvents, such as for example water, and/orcan be in the form of a fluid, for example in the form of a suspension,emulsion or solution.

A pharmaceutical composition according to the present invention can alsocomprise preservatives and other additives, such as for examplepreservatives and other additives selected from the group consisting ofantimicrobials, anti-oxidants, chelating agents, active agent(s) for usein the treatment and/or prevention of at least one of Graft-versus-Hostdisease and Aplastic Anemia or active agent(s) for promoting engraftmentafter haematopoietic stem cell transplantation and inert gases and thelike, and/or proteinaceous carriers, such as e.g. serum albumin orimmunoglobulin, in particular of human origin.

A pharmaceutical composition according to the present invention can beadministered to the subject at a suitable dose. The dosage regimen canbe for example determined by an attending physician. As well known inthe art, dosages for a patient can depend upon many factors, such as thepatient's size, body surface area, age, weight, administration forprevention or treatment purposes, target indication (Graft-versus-Hostdisease, Aplastic Anemia, etc.), the particular compound to beadministered, general health, and other drugs being administeredconcurrently. According to one embodiment, at least one antibody of thepresent invention (e.g. one or two or three or more antibodies of thepresent invention), in particular for use in the treatment and/orprevention of Graft-versus-Host disease, can be administered to apatient in an amount between 1 mg of said at least one antibody/m² bodysurface area per day and 4.5 mg of said at least one antibody/m² bodysurface area per day, in particular in an amount between 2 mg of said atleast one antibody/m² body surface area per day and 4.5 mg of said atleast one antibody/m² body surface area per day. In particular, anamount of about 2 mg of said at least one antibody/m² body surface areaper day or an amount of about 3 mg of said at least one antibody/m² bodysurface area per day or an amount of about 4.5 mg of said at least oneantibody/m² body surface area per day can be administered to a patient.The at least one antibody of the present invention may be present in theabove-mentioned amounts in a pharmaceutical composition. As used herein,the term “at least one antibody” encompasses one antibody or two or more(e.g. three or more) antibodies.

According to one embodiment, a pharmaceutical composition of the presentinvention can be a pharmaceutical composition which comprises anantibody mixture produced by hybridoma cell line deposit PD 12002. andoptionally at least one pharmaceutically acceptable excipient.

The pharmaceutical composition can—according to one embodiment—comprisethe antibodies produced by hybridoma cell line deposit PD 12002, inparticular for use in the treatment and/or prevention ofGraft-versus-Host disease for administration to a patient in an amountbetween 1 mg of said antibody mixture (produced by hybridoma cell linedeposit PD 12002)/m² body surface area per day and 4.5 mg of saidantibody mixture (produced by hybridoma cell line deposit PD 12002)/m²body surface area per day, in particular in an amount between 2 mg ofsaid antibody mixture (produced by hybridoma cell line deposit PD12002)/m² body surface area per day and 4.5 mg of said antibody mixture(produced by hybridoma cell line deposit PD 12002)/m² body surface areaper day. According to one embodiment, an amount of about 2 mg of saidantibody mixture/m² body surface area per day or an amount of about 3 mgof said antibody mixture/m² body surface area per day or an amount ofabout 4.5 mg of said antibody mixture/m² body surface area per day canbe administered to a patient. The amounts of: 2, 3, or 4.5 mg ofantibody mixture produced by hybridoma cell line deposit PD 12002/m²body surface area per day being successfully used in a clinical setting.According to a further embodiment, a pharmaceutical composition foradministration to patients comprises the antibody in an amount between0.1 and 10 mg/m² body surface area per day.

According to one embodiment, the administration of pharmaceuticalcompositions of the present invention is intravenous administration,e.g. intravenous infusion or intravenous injection. Optionallyadditionally at least one immunosuppressive drug, e.g. at least oneimmunosuppressive drug selected from the group consisting ofcorticosteroids, and cyclosporine (in particular cyclosporine A) can beadministered to the patient, together with the antibody or separately.

Body surface area (BSA) can be calculated according to any known method.For example, the body surface area (BSA) of a patient can be calculatedaccording to the Mosteller formula of BSA (m²)=([Height (cm)× Weight(kg)]/3600)^(1/2) (Mosteller R D., N Engl J Med 1987 Oct. 22;317(17):1098, which is incorporated herewith by reference) or accordingto the DuBois and DuBois formula of BSA (m²)=0.20247× Height(m)^(0.725)×Weight (kg)^(0.425) (DuBois D; DuBois E F., Arch Int Mad1916 17:863-71, which is incorporated herewith by reference). Accordingto a preferred embodiment, the Mosteller formula is used for calculatingthe body surface area (BSA) of a patient.

A pharmaceutical composition for use in the treatment of Aplastic Anemiacan comprise at least one antibody of the present invention, andoptionally at least one pharmaceutically acceptable excipient. Said atleast one antibody (e.g. one or two or more antibodies) of the presentinvention can be administered to a patient in an amount between 1 mg ofsaid at least one antibody/m² body surface area per day and 2 mg of saidat least one antibody/m² body surface area per day, in particular in anamount of about 2 mg of said at least one antibody/m² body surface areaper day for use in the treatment of Aplastic Anemia. The at least oneantibody of the present invention can be present in a pharmaceuticalcomposition in the above-mentioned amounts or ranges of amounts. Inparticular, the pharmaceutical compositions can be administered byintravenous administration, e.g. infusion or injection. Optionally,additionally at least one immunosuppressive drug, in particularcyclosporine A, can be administered to the patient, together with the atleast one antibody or separately. According to one embodiment, thepharmaceutical composition for use in the treatment of Aplastic Anemiacan comprise an antibody mixture produced by hybridoma cell line depositPD 12002, and optionally at least one pharmaceutically acceptableexcipient.

According to one embodiment, an antibody mixture produced by hybridomacell line deposit PD 12002 can be administered for use in the treatmentof Aplastic Anemia to a patient in an amount between 1 mg of saidantibody mixture (produced by hybridoma cell line deposit PD 12002)/m²body surface area per day and 2 mg of said antibody mixture (produced byhybridoma cell line deposit PD 12002)/m² body surface area per day, inparticular in an amount of about 2 mg of said antibody mixture (producedby hybridoma cell line deposit PD 12002)/m² body surface area per day.

Moreover, doses of an antibody of the present invention below or abovethe above indicated exemplary ranges can be administered, e.g. fortreating and/or preventing at least one of Graft-versus-Host disease andAplastic Anemia or for promoting engraftment after hematopoietic stemcell transplantation, especially considering the aforementioned factors.A pharmaceutical composition of the present invention can be formulatedto be short-acting, fast-releasing, long-acting, or sustained-releasing.

Furthermore, a pharmaceutical composition of the present invention cancomprise further biologically active agents, depending on the intendeduse of the pharmaceutical composition.

The pharmaceutical composition of the present invention can furthercomprise at least one immunosuppressive drug, in particular an effectiveamount of at least one immunosuppressive drug. The immunosuppressivedrug can be for example at least one drug selected from the groupconsisting of corticosteroids, in particular 6-methylprednisolone, andcyclosporine, in particular cyclosporine A. Moreover, the pharmaceuticalcomposition can comprise a combination of therapies where the secondactive ingredient is not included in the same composition as theanti-CD26 antibody.

In one embodiment, the pharmaceutical composition of the presentinvention (e.g. for treating and/or preventing at least one ofGraft-versus-Host disease and Aplastic Anemia or for promotingengraftment after hematopoietic stem cell transplantation) comprising atleast one antibody of the present invention, in particular an antibodymixture of the present invention, can comprise one or morecorticosteroids, one or more antihistamines, water and sodium chloride.In particular, the pharmaceutical composition can further comprise oneor more corticosteroids, one or more antihistamines and saline, e.g.physiological saline (especially saline containing about 0.9% w/v NaCl).In particular, these aqueous pharmaceutical compositions can beadministered by infusion, especially slow infusion, in particular byintravenous administration. The at least one antibody of the presentinvention, in particular the antibodies produced by hybridoma cell linedeposit PD 12002, one or more corticosteroids, and one or moreantihistamines, each alone or in combination, can be preferably presentin a therapeutically effective amount. In particular, the pharmaceuticalcomposition can comprise the at least one antibody of the presentinvention in amount between 1 mg of said at least one antibody/m² bodysurface area per day and 4.5 mg of said at least one antibody/m² bodysurface area per day for use in the treatment and/or prevention ofGraft-versus-Host disease or can comprise for use in the treatmentand/or prevention of Aplastic Anemia at least one antibody of thepresent invention in an amount between 1 mg of said at least oneantibody/m² body surface area per day and 2 mg of said at least oneantibody/m² body surface area per day. Moreover, the pharmaceuticalcomposition can comprise currently preferred ranges of amounts andamounts of at least one antibody of the present invention, in particularthe antibody mixture as discussed herein. Moreover, the pharmaceuticalcomposition can comprise a combination of therapies where the secondactive ingredient is not included in the same composition as theanti-CD26 antibody.

Corticosteroids are well known in the art and can comprise in particularmineralocorticoids and glucocorticoids. Glucocorticoids can beanti-inflammatory agents. As used herein, the term corticosteroids caninclude steroids which can be in particular produced in the adrenalcortex of vertebrates, as well as can encompass syntheticcorticosteroids or synthetic or natural corticosteroid analogs,including compounds that mimic the activity of natural steroid hormones,such as e.g. cortisone and hydrocortisone. Corticosteroid analogs may inparticular encompass synthetic or natural chemical compounds whichresemble in structure and/or function any of naturally occurringsteroids elaborated by the adrenal cortex.

One or more corticosteroids can be selected from the group consisting ofalclometasone dipropionate, amcinonide, amcinafel, amcinafide,beclamethasone, betamethasone, betamethasone dipropionate, betamethasonevalerate, clobetasone propionate, chloroprednisone, clocortelone,cortisol, cortisone, cortodoxone, difluorosone diacetate, descinolone,desonide, defluprednate, dihydroxycortisone, desoximetasone,dexamethasone, deflazacort, diflorasone, diflorasone diacetate,dichlorisone, esters of betamethasone, fluazacort, flucetonide,flucloronide, fludrotisone, fluorocortisone, flumethasone, flunisolide,fluocinonide, fluocinolone, fluocinolone acetonide, flucortolone,fluperolone, fluprednisolone, fluroandrenolone acetonide, fluocinoloneacetonide, flurandrenolide, fluorametholone, fluticasone propionate,hydrocortisone, hydrocortisone butyrate, hydrocortisone valerate,hydrocortamate, loteprendol, medrysone, meprednisone, methylprednisone,methylprednisolone, 6-methylprednisolone, mometasone furoate,paramethasone, paramethasone acetate, prednisone, prednisolone,prednidone, prednicarbate, triamcinolone acetonide, triamcinolonehexacatonide, tixocortol prednisolone, and triamcinolone,pharmaceutically acceptable salts thereof, derivatives thereof, andmixtures thereof.

Antihistamines are known in the art and can be in particularpharmaceutical drugs that can reduce or counteract the action ofhistamine. In particular, an antihistamine can be a H₁-receptorantagonist.

One or more antihistamine drugs can be in particular selected from thegroup consisting of astemizole, azelastine, buclizine, brompheniramine,chlorpheniramine, cetirizine, clemastine, cyclizine, desloratidine,dexbrompheniramine, diphenhydramine, doxylamine, ebastine, emedastine,epinastine, fexofenadine, hydroxyzine, ketotifen, levocabastine,levocetirizine, loratidine, mequitazine, mizolastine, olopatadine,oxatomide, phenindamine, pheniramine, pyrilamine, terfenidine,triprolidine, pharmaceutically acceptable salts, isomers or prodrugsthereof.

According to another aspect the present invention provides a kitcomprising: (i) at least one antibody of the present invention, inparticular an antibody mixture of the invention or a compositioncomprising an antibody mixture of the present invention; andadditionally (ii) a) at least one immunosuppressive drug, e.g. at leastone immunosuppressive drug selected from the group consisting ofcorticosteroids, and cyclosporine, in particular cyclosporine A or b) atleast one corticosteroid and at least one antihistamine. The at leastone antibody can comprise or consist of the antibodies produced byhybridoma cell line deposit PD 12002. In particular, the kit cancomprise the at least one antibody of the present invention in amountbetween 1 mg of said at least one antibody/m² body surface area per dayand 4.5 mg of said at least one antibody/m² body surface area per dayfor use in the treatment and/or prevention of Graft-versus-Host diseaseor can comprise for use in the treatment and/or prevention of AplasticAnemia at least one antibody of the present invention in an amountbetween 1 mg of said at least one antibody/m² body surface area per dayand 2 mg of said at least one antibody/m² body surface area per day.Moreover, the pharmaceutical composition can comprise currentlypreferred ranges of amounts and amounts of at least one antibody of thepresent invention as discussed herein.

The kit can be a kit for use as a medicament, in particular a kit forpreventing and/or treating Graft-versus-Host disease (GvHD), preferablyafter haematopoietic stem cell transplantation, and/or a kit for use intreating Aplastic Anemia, preferably Severe Aplastic Anemia, and/or akit for promoting engraftment after haematopoietic stem celltransplantation. The terms kit of parts and kit are used interchangeablyherein.

In vivo and in vitro production of antibodies of the invention intransgenic animals, obtained by genetic manipulation of non-humananimals, in particular non-human mammals, using at least one of thenucleotide sequences described in the present invention by methods knownto a skilled person, is also comprised within the scope of the presentinvention.

In the following, the present invention will be described in more detailwith reference to the following non-limiting examples. It is, however,understood that the present invention is not limited to the followingexamples.

EXAMPLES Example 1 Determining the Nucleotide Sequences

This process can be summarized into three phases:

Phase I—Cloning of Genes Encoding for Antibody Chains

-   -   Extraction of total RNA from Hybridoma cells producing CDina26        (Hybridoma cell line deposit under the reference PD 12002, see        above);    -   Reverse transcription;    -   Amplification of the gene of interest using specific        oligonucleotides as primers;    -   Cloning of gene using a prokaryotic vector;    -   Bacterial transformation;    -   Control of cloning procedure, selecting suitable transformed        clones.        Phase II—Sequencing of Genes Encoding for Antibody Variable        Chains and Bioinformatics Analysis:    -   Picking and sequencing of 100 colonies for both VH and VL        chains;    -   Bioinformatics analysis of sequences;    -   Generation of a consensus sequence, if required, for the VL and        VH chains and indication of the consensus sequence differing        bases and their percentage.        Phase III—Sequencing of Genes Encoding for Antibody Constant        Chains:    -   Sequencing of the corresponding CL chain for VL chain;    -   Sequencing of corresponding CH1-CH2-CH3 chain for VH chain.

The results show the presence in mRNA samples of three groups of VLchain sequences (VL group 1, VL group 2 and VL group 3) and two groupsof VH chain sequences (VH group 1 and VH group 2).

Determining the Amino Acid Sequence

Mass spectrometry analysis and N-terminal sequencing were used toconfirm the amino acid sequence present in CDina26 sample. In CDina26sample was confirmed the presence of VL group 1 and its related CLsequence, of VL group 3 chain and of VH group 1 related to itsCH1-CH2-CH3 chain. Neither VL Sequences group 2 nor VH sequences group 2have been detected as aminoacidic sequences in the CDina26 antibodysample.

Example 2 CDina26 Binding to Human and Porcine CD26

The binding of CDina26 to human CD26 has been compared to its binding tominipig CD26 by Biacore® assay and multiparametric flow cytometry.CDina26 is not able to recognize Porcine Antigen neither as solubleprotein or as a transmembrane protein expressed on T lymphocytes.

-   1. Flow cytometry analysis was utilized to determine the ability of    CDina26 to bind to cell surface expressed CD26.    -   Lymphocytes were isolated from human or minipig peripheral blood        samples by centrifugation on gradient density. The Binding of        CDina26 to human T Lymphocytes was analyzed by multiparametric        flow cytometry.    -   More than 45% of Human T lymphocytes expressing CD3 marker bind        to CDina26. On the contrary only 2% of porcine T lymphocytes        expressing CD3 marker bind to CDina26. This may be seen from        FIG. 9.-   2. Binding of CDina26 to either Human or Porcine Antigen (purchased    from Sigma CAT# D4943 and D7052) was analysed by Biacore®.    -   CDina26 was captured on the matrix by a polyclonal anti-mouse Ig        antibody previously immobilized on a flow cell of Biacore® T100        sensor chip. Either Human or Porcine Antigen were injected in        solution over the immobilized CDina26.    -   Human antigen, injected at two concentrations over immobilized        CDina26, gave a dose dependent response with a high affinity        binding, indicated by a kinetic dissociation rate of 2.8×10⁻⁵        s⁻¹.    -   Porcine antigen, injected over immobilized CDina26, gave no        binding.

FIG. 11 shows binding of Human (left) and Porcine (right) Antigen (Ag)on captured CDina26, measured in Biacore® Resonance Units (RU).

Example 3 Antibody Production

CDina26 is produced by hybridoma cell line deposit at CBA-ICLC of Genoa(Italy) under reference PD 12002. The hybridomas may be cultured inserum free medium. CDina26 comprises a mixture of murine antibodies. Inparticular, CDina26 comprises a murine, monoclonal antibody, which is ofIgG 2B class and specifically binds to human CD26.

A pharmaceutical composition comprising CDina26 is present in the formof a clear colourless solution containing CDina26, which solution can beused for intravenous infusion (e.g. 1 mg of CDina26/1 ml of solution;the solution can be contained in a vial).

Example 4 Pharmacodynamic Study In Vitro

In vitro pharmacodynamics studies were performed for thecharacterization of murine monoclonal antibody against CD26, inparticular for the characterization of the antibodies produced by PD12002 hybridoma cell line deposit, referred to herein as CDina26.

The aim of these studies was to evaluate the expression of and specificbinding to CD26 on the surface of cell population involved in the immuneresponse, in particular

-   -   T lymphocytes    -   B lymphocytes    -   NK (natural killer) cells    -   Monocytes    -   Dendritic cells

Example 4a

CDina26 antigen expression was evaluated in resting T, B and NK cells(T₀) purified from 5 healthy donors (corresponding to “ESP.1” to “ESP.5”in Table 1) and then in cells activated via allogeneic stimuli (mixedlymphocyte culture (MLC)), mitogeneic stimuli (phytohemagglutinin (PHA))or antigenic stimuli (Candida albicans).

In the present application, the abbreviation MFIR is used asabbreviation for mean Relative Fluorescence Intensity as known in theart.

TABLE 1 Evaluation of percentage of CDina26 + cells and MFIR indexwithin lymphocyte subpopulations in healthy donors before and aftermitogenic stimuli with PHA. % CD26 MFIR ESP. 1 PHA subpopulations TCD3+/CD4+ 86→84 D 5→ 3 D T CD3+/CD8+ 75→67 D  6→14 A T CD3+/CD16+ 16→61A 1→ 6 A T CD3+/CD56+ 36→68 A 4→ 7 A NK CD3−/CD16+ 11→58 A 0→ 1 A NKCD3−/CD56+ 10→32 A 0→ 1 A B CD19+/CD20+ 13→80 A  2→10 A ESP. 2 PHAsubpopulations T CD3+/CD4+ 74→91 A 10→ 9  D T CD3+/CD8+ 57→93 A 15→15  TCD3+/CD16+ 16→85 A 2→ 6 A T CD3+/CD56+ 38→97 A  6→18 A NK CD3−/CD16+15→50 A 2→ 1 D NK CD3−/CD56+ 10→38 A 1→ 1 B CD19+/CD20+ 11→95 A  2→11 AESP. 3 PHA subpopulations T CD3+/CD4+ 75→82 A 8→ 7 D T CD3+/CD8+ 45→82 A4→ 9 A T CD3+/CD16+ 15→69 A 1→ 6 A T CD3+/CD56+ 41→92 A 4→ 9 A NKCD3−/CD16+  7→14 A 1→ 2 A NK CD3−/CD56+  6→25 A 1→ 3 A B CD19+/CD20+14→92 A  8→10 A ESP. 4 PHA subpopulations T CD3+/CD4+ 81→83 A  3→10 A TCD3+/CD8+ 53→91 A  2→13 A T CD3+/CD16+ 23→96 A  1→18 A T CD3+/CD56+52→94 A  3→14 A NK CD3−/CD16+ 23→67 A 1→ 6 A NK CD3−/CD56+ 14→45 A 0→ 4A B CD19+/CD20+ 12→92 A  0→14 A ESP. 5 PHA subpopulations T CD3+/CD4+74→92 A 30→10  D T CD3+/CD8+ 48→92 A 11→12  A T CD3+/CD16+  9→81 A  2→13A T CD3+/CD56+ 15→91 A 14→11  D NK CD3−/CD16+  8→12 A 2→ 5 A NKCD3−/CD56+  3→20 A 2→ 3 A B CD19+/CD20+  3→90 A  2→10 A In the columnsentitled “% CD26” and “MFIR”, respectively, the first value correspondsto T0, the second value after the arrow corresponds to the stimulatedcells: A, increase in value or a percentage of expression (MFIR) D,decrease in value or a percentage of expression (MFIR) T0 being anabbreviation for Time 0, i.e. for the point in time preceding the cellstimulation.

As may be seen from Table 1, CD26 expression on activated lymphocytesshowed an increased percentage of CD26+ cells after stimulation with PHAas compared to T₀ with the exception of T CD3+CD4+ and T CD3+CD8+ inexperiment number 1 (“ESP 1”). Level of expression (MFRI index value)varied among the different T, B and NK subpopulations and the 5 healthyvolunteers. Furthermore, mitogeneic stimuli appears to modestly increasethe values of MFIR (Table 1).

Example 4b

The analysis of the results reported in Table 2 showed that in allsubsets analyzed, with the exception of CD3+CD4+T subpopulations of allhealthy volunteers, there is an increase in the percentage oflymphocytes expressing CDina26 antigen after antigenic stimuli.

In all experiments, except one (“ESP.5”), there is an increase of MFIRvalues. It may be also noted that the increase of MFIR values ascompared with time 0 is greater in cultures stimulated with Candida thanthose stimulated with PHA. The Candida antigen stimulation significantlyincreases the expression of CD26 molecule on the cell membrane,especially in subpopulations T CD3+CD16+ and T CD56+CD3+.

TABLE 2 Evaluation of percentage of CDina26 + cells and MFIR indexwithin lymphocyte subpopulations in healthy donors before and afterantigenic stimuli with Candida albicans. % CD26 MFIR ESP. 1 Candidasubpopulations T CD3+/CD4+ 86→71 D 5→ 8  A T CD3+/CD8+ 75→80 A 6→14 A TCD3+/CD16+ 16→46 A 1→16 A T CD3+/CD56+ 36→68 A 4→13 A NK CD3−/CD16+11→11 0→ 4  A NK CD3−/CD56+ 10→32 A 0→ 4  A B CD19+/CD20+ 13→21 A 2→ 3 A ESP. 2 Candida subpopulations T CD3+/CD4+ 74→64 D 10→11  A T CD3+/CD8+57→78 A 15→29  A T CD3+/CD16+ 16→98 A 2→36 A T CD3+/CD56+ 38→99 A 6→38 ANK CD3−/CD16+ 15→97 A 2→23 A NK CD3−/CD56+ 10→91 A 1→19 A B CD19+/CD20+11→26 A 2→ 5  A ESP. 3 Candida subpopulations T CD3+/CD4+ 75→60 D 8→14 AT CD3+/CD8+ 45→59 A 4→15 A T CD3+/CD16+ 15→30 A 1→89 A T CD3+/CD56+41→71 A 4→85 A NK CD3−/CD16+  7→26 A 1→ 9  A NK CD3−/CD56+  6→19 A 1→ 7 A B CD19+/CD20+ 14→14 8→ 5  D ESP. 4 Candida subpopulations T CD3+/CD4+81→81 3→22 A T CD3+/CD8+ 53→65 A 2→24 A T CD3+/CD16+ 23→90 A 1→76 A TCD3+/CD56+ 52→94 A 3→79 A NK CD3−/CD16+ 23→79 A 1→46 A NK CD3−/CD56+14→88 A 0→58 A B CD19+/CD20+ 12→15 A 0→6  A ESP. 5 Candidasubpopulations T CD3+/CD4+ 74→4  D 30→ 9   D T CD3+/CD8+ 48→65 A 11→20 A T CD3+/CD16+  9→87 A 2→39 A T CD3+/CD56+ 15→94 A 14→41  A NKCD3−/CD16+  8→91 A 2→34 A NK CD3−/CD56+  3→87 A 2→30 A B CD19+/CD20+  3→6 A 2→11 A In the columns entitled “% CD26” and “MFIR”, respectively,the first value corresponds to T0, the second value after the arrowcorresponds to the stimulated cells: A, increase in value or apercentage of expression (MFIR) D, decrease in value or a percentage ofexpression (MFIR)

Example 4c

Table 3 shows that in all experiments, the percentage of lymphocytespositive for CDina26 antigen increased in all lymphocyte subpopulationspresent after stimulation with mixed lymphocyte culture with theexception of CD3+CD4+T subpopulation.

TABLE 3 Evaluation of percentage of CDina26 + cells and MFIR indexwithin lymphocyte subpopulations in healthy donors before and afterallogeneic stimuli with MLC (mixed lymphocyte culture). % CD26 MFIR ESP.1 MLC subpopulations T CD3+/CD4+ 86→67 D 5→ 6  A T CD3+/CD8+ 70→75 A 6→8  A T CD3+/CD16+ 16→83 A 1→ 9  A T CD3+/CD56+ 36→89 A 4→11 A NKCD3−/CD16+ 11→74 A 0→ 5  A NK CD3−/CD56+ 10→70 A 0→ 5  A B CD19+/CD20+13→19 A 2→ 3  A ESP. 2 MLC subpopulations T CD3+/CD4+ 74→63 D 10→10  TCD3+/CD8+ 57→75 A 15→18  A T CD3+/CD16+ 16→86 A 2→19 A T CD3+/CD56+38→95 A 6→18 A NK CD3−/CD16+ 15→17 A 2→11 A NK CD3−/CD56+ 10→84 A 1→ 9 A B CD19+/CD20+ 11→11 2→ 2  ESP. 3 MLC subpopulations T CD3+/CD4+ 75→57D  8→ 18 A T CD3+/CD8+ 45→53 A  4→ 25 A T CD3+/CD16+ 15→48 A  1→ 67 A TCD3+/CD56+ 41→77 A  4→166 A NK CD3−/CD16+  7→51 A 1→13 A NK CD3−/CD56+ 6→47 A 1→13 A B CD19+/CD20+ 14→25 A 8→22 A ESP. 4 MLC subpopulations TCD3+/CD4+ 81→76 D 3→12 A T CD3+/CD8+ 53→78 A 2→18 A T CD3+/CD16+ 23→83 A1→16 A T CD3+/CD56+ 52→89 A 3→18 A NK CD3−/CD16+ 23→87 A 1→10 A NKCD3−/CD56+ 14→79 A 0→11 A B CD19+/CD20+ 12→27 A 0→ 6  A ESP. 5 MLCsubpopulations T CD3+/CD4+ 74→62 D 30→11  D T CD3+/CD8+ 48→68 A 11→13  AT CD3+/CD16+  9 →85 A 2 →19  A T CD3+/CD56+ 15→93 A 14→24  A NKCD3−/CD16+  8 →85 A 2 →13  A NK CD3−/CD56+  3 →85 A 2 →15  A BCD19+/CD20+  3 →28 A 2 →11  A In the columns entitled “% CD26” and“MFIR”, respectively, the first value corresponds to T0, the secondvalue after the arrow corresponds to the stimulated cells: A, increasein value or a percentage of expression (MFIR) D, decrease in value or apercentage of expression (MFIR)

The MFIR index increased after stimulation as compared with time 0 inall subsets and all experiments, with the exception of the subpopulationof CD4+CD3+T in experiments number 2 (“ESP.2”) and number 5 (“ESP.5”).

Similar to the stimulation with Candida, the allogeneic stimulationconstantly increases the expression of CDina26 antigen and MFIR index onmost lymphocyte subpopulations. A significant increase was observedmainly in subpopulations of CD16+CD3+T and CD56+CD3+T experiment number3 (“ESP.3”).

Example 4d

Finally, the expression of CDina26 antigen was investigated on leukocytesubpopulations in patients after allo-HSCT. In particular, two patientswere assessed during the early tests for monitoring immunereconstitution post-transplant, while four patients who developed acuteGvHD, it was possible to carry out the assessment both during the onsetof GvHD and after the resolution. Compared with controls, patientsshowed a highly variable distribution of T, NK and B subpopulations(Table 4), which is compatible with the process of haematopoieticreconstitution that occurs in the months following transplantation.

TABLE 4 Distribution (measured as %) of lymphocyte subpopulations in 6patients who underwent HSCT evaluated at the time of immunereconstitution and compared with the range obtained by evaluating 5healthy donors. Percentage (%) Healthy donors Patients Subpopulationrange CK IP BGL* BG* CJ* DF* T CD3+/CD4+ 44-58 6 12 22 6 7 12 TCD3+/CD8+ 20-30 69 40 42 50 7 10 T CD3+/CD16+  5-10 3 4 17 9 4 10 TCD3+/CD56+  4-10 2 10 11 2 2 13 NK CD3−/CD16+ 2-9 14 18 13 8 52 34 NKCD3−/CD56+  7-15 18 17 1 8 67 67 B CD20+/CD19+ 3-7 0 7 0 1 0 5 *At thetime of evaluation, the patients had developed acute GvHD grade II.

Analyzing the percentage of CD26 (Table 5) in all patients there was anincrease of the expression of this molecule within subpopulations TCD16+CD3+ and T CD56+CD3+ and NK compared to the control range.

TABLE 5 Distribution (measured as %) of CDina26 + cells in lymphocytesubpopulations in 6 patients who underwent HSCT evaluated at the time ofimmune reconstitution and compared with the range obtained by evaluating5 healthy donors. % CD26 Healthy donors Patients Subpopulation range CKIP BGL* BG* CJ* DF* T CD3+/CD4+ 74-86  58 58 90 60 86 97 T CD3+/CD8+45-75  29 22 68 25 74 93 T CD3+/CD16+ 9-23 30 53 90 15 82 96 TCD3+/CD56+ 15-52  54 47 100 90 86 95 NK CD3−/CD16+ 7-23 33 21 33 17 6149 NK CD3−/CD56+ 3-14 36 25 37 19 74 54 B CD20+/CD19+ 3-14 0 36 0 0 0 93*At the time of evaluation, the patients had developed acute GvHD gradeII.

In patients who developed aGvHD, in some cases, the value of thepercentage is higher than in the two patients free of this complication(CK, IP). The patient DF showed significantly higher percentages of CD26in all subsets, an index of cellular activation was very pronounced andit was not possible to estimate the index of the molecule CD26 MFIR.

In the two patients who did not develop aGvHD, the index showed MFIRvalues falling in the range of control, while in three patients withaGvHD in progress, MFIR values were increased in almost all subsets, andmost especially in the sub-T CD3+CD16+ and CD3+T CD56+(Table 6).

TABLE 6 Distribution (measured as MFIR) of CDina26 + cells in lymphocytesubpopulations in 6 patients who underwent HSCT evaluated at the time ofimmune reconstitution and compared with the range obtained by evaluating5 healthy donors. Healthy MFIR index donors Patients Subpopulation rangeCK IP BGL* BG* CJ* DF* T CD3+/CD4+  3-30 3 1 62 25 21 n.a T CD3+/CD8+ 2-15 1 2 25 7 23 n.a T CD3+/CD16+ 1-2 2 1 89 9 23 n.a T CD3+/CD56+ 3-14 4 2 165 37 40 n.a NK CD3−/CD16+ 0-2 1 1 5 5 7 n.a NK CD3−/CD56+0-2 1 1 6 3 8 n.a B CD20+/CD19+ 0-8 0 1 0 0 0 n.a n.a: not applicable

Example 4e

The expression of CD26 was also studied on mesenchymal stem cells,endothelial cells and fibroblasts.

Three experiments were set up using Mesenchymal Stem Cells (MSCs),propagated in vitro derived from bone marrow of three healthy adults.The analysis by flow cytometry showed that cells positive for CD13 andCD73, markers characteristic of MSCs are, however, negative for theexpression of CD26 (CDina26 antigen). Three experiments were set upusing endothelial cells (DC) from umbilical cord of three healthysubjects. Endothelial cells are negative for the expression of CD26(CDina26 antigen).

Three experiments were set up using skin fibroblasts derived from threehealthy adults. The analysis by flow cytometry demonstrated that 40% offibroblasts expressed CD26 (range 32%-45%) with a MFIR index from 4 to13.

Three experiments were set up using dendritic cells differentiated invitro. Flow cytometric analysis showed that the cells positive for CD1a,a marker characteristic of DC differentiated in vitro, are however,negative for the expression of CD26.

The flow cytometry showed that monocytes cells positive for CD14 arepositive for the expression of CD26 (range 97%-100%), MFIR indexvariable from 1 to 17.

These data demonstrated that T and NK subpopulations increased both thepercentage or the expression of CDina26 antigen (MFIR) on membranesurface.

In aGvHD patients an increased expression of CD26 can be observed in TCD3+CD16+, T CD3+CD56+ and NK as compared with healthy donors.

These data summarize the ability of monoclonal antibodies anti CD26 ofCDina26 to specifically bind to activated regulatory T cells,interfering with their expansion and with their role in the modulationof the immune response in aGvHD.

Example 5 Clinical Studies Treatment of Graft-Versus-Host Disease (GvHD)

A clinical study has been conducted for establishing the safety andefficacy of CDina26 in aGvHD (acute Graft-versus-Host Disease) patient.

SUMMARY

Patients enrolled in the study received a fixed dose of CDina26 of 2mg/day (which corresponds to an average of 1.11 mg/m² per day) for 5consecutive days. The composition administered to patients included theantibody of the present invention in a range between 2 and 10 mgaccording to body surface diluted in 100 ml of sterile saline solutiontogether with corticosteroids and antihistamine. Patients continued toreceive their standard GvHD treatment (6-methylprednisolone 1-2mg/kg/day i.v., and cyclosporine). Supportive care was the conventionalantibacterial, antifungal and antiviral therapy. In the presentapplication, the units mg/m² and mg/(m² body surface area) are usedinterchangeably.

The main evaluation of this study was the frequency of patients“responding” to the studied treatment, assessed on day 10 after 5 daysof therapy.

The definition of responsiveness was based on the following criteria:

-   -   COMPLETE RESPONSE (CR)→resolution of all signs of GvHD    -   PARTIAL RESPONSE (PR)→improvement in grading of GvHD    -   STABILITY→no change in grading of GvHD    -   AGGRAVATION→aggravation in grading of GvHD

Patients with complete or partial response were considered responsive.

TABLE 7 Glucksberg acute GvHD staging (Glucksberg et al., 1974) OrganStage 1 Stage 2 Stage 3 Stage 4 Skin <25% of body 25-50% of bodyGeneralized Generalized erythroderma Maculo-papular surface surfaceerythroderma with bullae formation rash and desquamation Liver 2-3 mg/dl3.1-6 mg/dl 6.1-15 mg/dl >15 mg/dl Bilirubin GI >500 ml/day >1000ml/day >1500 ml/day >1500 ml/day Diarrhoea Severe abdominal pain with orwithout ileus

TABLE 8 Glucksberg of acute GVHD grading (Glucksberg et al., 1974)Overall grade Skin Liver GI ECOG I 1-2 0 0 0 II 1-3 1 and/or 1   0-1 III2-3 2-4 and/or 2-3 2-3 IV 2-4 2-4 and/or 2-4 3-4 GI: gastrointestinaltract Eastern Cooperative Oncology Group (ECOG) Performance Status:Additional evaluations include the following: staging of GvHD, organ fororgan (This evaluation is carried out at the day +10 and day +30.);complications occurrence such as: infection, bleeding, transfusionnecessity (This evaluation is carried out until the end of thehospitalization at day +30.); at the 1 year visit the followingparameters are recorded: survival status, Karnofsky index, chronic GvHD,possible relapse of the hematological disease for which transplantationwas performed, possible appearance of a new tumor.Demographic and Other Baseline Characteristics

Eleven patients were included in the data set for the efficacyevaluation. Seven patients had grade III aGvHD, one patient had grade IVGvHD and one patient had grade II GvHD.

The median time to onset of aGvHD was 10 days (range 4-73 days).

All these patients had aGvHD which was considered at high risk becauseof visceral organ involvement.

Efficacy evaluation: the frequency of patients “responding” to 2 mg ofCDina26 i.v. daily, administered for five consecutive days, at thecurrent median follow up of 433 days is 11 on 12 (90%): six (6) completeresponses, five (5) partial responses and one (1) non response.

Following the data in four patients (pt. 01, 03, 12, 14) in Glucksbergacute GvHD grading. The following abbreviations are used in thefollowing Table:

pre CDina26 stands for patients' GvHD grade before the treatment withCDina26; best CDina26 stands for best patients' GvHD grade value afterthe treatment with CDina26; final CDina26 stands for the most recentpatients' GvHD grade value; The Follow up values represent the daysafter CDina26 treatment.

TABLE 9 Pre CDina26 Best CDina26 Final CDina26 GvHD GvHD GvHD GvHD GvHDGvHD GvHD GvHD GvHD GvHD GvHD GvHD Follow ID Skin Liver gut grade SkinLiver gut grade Skin Liver gut grade Up Response Pt01 2 0 2 3 0 0 0 0 00 0 0 365 CR Pt03 2 0 3 3 0 0 0 0 2 0 0 1 365 PR Pt12 2 0 0 2 0 0 0 0 10 1 1 341 PR Pt14 0 0 2 2 0 1 0 0 0 1 0 0 208 CR

FIG. 4a shows Grading of Skin GvHD, day 1, 10, 30 and last day (“last”in FIG. 4a ) of the patient in the study. FIG. 4b illustrates Grading ofliver GvHD, day 1, 10, 30 and last day of the patient in the study. FIG.4c shows Grading of gut GvHD, day 1, 10, 30 and last day of the patientin the study.

Immune Recovery

Immunodeficiency is common in patients with acute GvHD, especially afterprolonged treatment with steroids, and infections are the consequence ofsevere combined immune deficiency. FIG. 5 outlines absolute CD4 countsin 6 patients. CD4 counts tend to increase or remain essentially stableafter CDina26 therapy, rather than showing a decline, as would beexpected in case of a strong cytolytic activity of the antibody. Theunit “counts/ul” stands for counts per microliter of blood of a patient.

Brief Summary of Adverse Events

The safety of CDina26 has been examined through a review of adverseevents. Serious toxicities involving the haematologic and respiratorysystems were considered as expected consequences of the conditioningregimen and transplant process. Overall 8 serious and 26 non-seriousadverse events were reported as not related to CDina26 treatment. TheMost frequently reported adverse events were related to the SOCs (SystemOrgan Classes) “Infections and infestations” (n=5), “Renal and urinarydisorders” (n=5), “Respiratory, thoracic and mediastinal disorders”(n=5) and “Skin and subcutaneous tissue disorders” (n=4).

Infections are a frequent complication of acute GvHD and steroidtherapy. Therefore it was not unexpected to see a number of infectiousepisodes in the patients.

Eight fatal adverse events occurred after Day +100. None of these wasconsidered as related to the treatment with CDina26.

Due to the fact that Steroid-resistant acute Graft-versus-Host-Diseasefollowing allogeneic haematopoietic stem cell transplantation isassociated with a high mortality rate, the transplant related mortality(deaths due to transplant related complications, the majority of whichare associated with aGvHD) was evaluated.

Considering all patients treated with CDina26 the incidence ofTransplant Related Mortality (TRM) after 6 months from the treatment is25% (3 of 12). Among TRM, two patients died from GvHD. None of these wasrelated to treatment with CDina26.

FIG. 6 outlines the projected cumulative incidence of transplant relatedmortality of 13 control patients with grade III-IV acute GvHD, treatedwith steroids, cydosporine and other immunosuppressive drugs, comparedwith 9 patients treated with steroids cyclosporine and CDina26.

The cumulative incidence of transplant related mortality in the 9patients receiving CDina26 is currently 12%, compared to 62% for matchedcontrols not receiving CDina26 (p=0.02).

FIG. 7 outlines the projected actuarial Survival of 13 control patientswith grade III-IV acute GvHD, treated with steroids, cydosporine andother immunosuppressive drugs, compared with 9 patients treated withsteroids, cyclosporine and CDina26. The p value is 0.2.

In conclusion, CDina26 patients show a lower mortality than non-CDina26patients, i.e. patients that were not treated with CDina26.

Conclusions

Results of murine monoclonal antibody against CD26 therapy for steroidrefractory acute GvHD in 11 patients have been extremely encouraging,both in terms of response and survival. Given the lack of any effectivetherapeutic measure in these circumstances, and given the lack of anyimprovement in therapy and outcome over the past 3 decades CDina26resulted in a high rate of responses and a high proportion of survivingpatients. It has to be pointed out that transplant mortality (TRM)directly attributed to the transplant and its complication is 62% forcontrol patients with GvHD III-IV not receiving murine monoclonalantibody against CD26, and 25% for patients receiving CDina26. Webelieve these results are very promising for the clinician.

Example 6

A pharmaceutical composition containing at least one antibody of thepresent invention, in particular containing the antibodies produced bythe PD 12002 hybridoma cell line deposit, CDina26, has for example thefollowing composition:

TABLE 10 Quantity Component per vial at least one antibody of thepresent invention, in 1 mg particular the antibodies produced by PD12002 hybridoma cell line deposit, CDina26 DPBS (Dulbecco's PhosphateBuffered Saline) 1 ml 1 ml of DPBS comprising: KCl 0.2 mg KH₂PO₄ 0.2 mgNaCl 8 mg Na₂HPO₄ × 7H₂O 2.16 mg Water for injection To 1 ml

This pharmaceutical composition can be administered in particular byintravenous injection.

Example 7 Treatment of SAA (Severe Aplastic Anemia)

One patient with acquired SAA developed pancytopenia after an allogeneicHSCT. The patient had mixed CD3 chimerism (37% autologous) suggestingpersistence of the auto-aggressive T cells, causing aplasia. Donorchimerism on bone marrow cells was 100% donor.

The patient received a course of anti-CD26 monoclonal antibodiesCDina26, 2 mg/day i.v. (2 mg of CDina26 provided in the form of asolution for intravenous administration) for 5 days, as an outpatient,in a Day Hospital. Treatment was well tolerated with no adverse effects.

Hb WBC Pt The blood counts of the patient after treatment are asfollows: 120 days before treatment 10 1.7 70 7 days before treatment 8.91.6 12 During five days the treatment with anti-CD26 antibodies wascarried out. 6 days after treatment 8.4 2.6 22 20 days after treatment7.6 2.5 22 31 days after treatment 10.4 4.8 32 48 days after treatment9.2 3.9 42 87 days after treatment 9.7 4.4 66

(Blood counts were obtained 87 days after treatment, i.e. nearly 3months after treatment, with anti-CD26 antibody). The followingabbreviations was used: Hb (Hemoglobin), WBC (White Blood Cells), Pt(Platelet). The data above show that an inhibition of CD26 can bebeneficial in patients with acquired SAA, due to its immunomodulatoryeffect, and the role in stem cell homing.

Example 8 Mapping of the PD 12002 Epitope

The epitopes recognized by the CDina26 antibody produced by hybridomacell line deposited as PD 12002 have been identified by CLIPS™ EpitopeMapping technology. See, e.g., U.S. Pat. No. 7,863,239 and U.S. Pat. No.7,972,993, the disclosures of which are herein incorporated by referencein their entirety. Briefly, CLIPS™ technology structurally fixespeptides into defined three-dimensional structures. This results infunctional mimics of even the most complex binding sites. The CLIPS™reaction takes place between bromo groups of the CLIPS™ scaffold andthiol side chains of cysteines. The reaction is fast and specific undermild conditions (Timmerman at al., J. Mol. Recognit. 2007; 20: 283-29).

CLIPS™ library screening starts with the conversion of the human CD26target protein into a library of overlapping peptide constructs, using acombinatorial matrix design. On a solid carrier, a matrix of linearpeptides is synthesized, which are subsequently shaped into spatiallydefined CLIPS™ constructs. Constructs representing several parts of adiscontinuous epitope in the correct conformation bind the antibody withhigh affinity, which is detected and quantified. Constructs presentingthe incomplete epitope bind the antibody with lower affinity, whereasconstructs not containing the epitope do not bind at all. Affinityinformation is used in iterative screens to define the sequence andconformation of epitopes in detail.

First, the adenosine deaminase binding domain (residues 356 to 522 ofSEQ ID NO: 144) of the human CD26 protein sequence was selected forin-depth analysis. This region of CD26 was extended and split into twooverlapping domains, that of residues 260 to 400, and 380 to 538 of SEQID NO: 144. Competitive binding assays revealed that CDina26 recognizesan epitope localized close to residue R358 of human CD26 (SEQ ID NO:144).

Second, a total of 5833 overlapping peptides of CD26 were synthesizedand tested for specific binding by CDina26. The analysis of linearpeptides identified multiple regions that were specifically recognizedby CDina26. Four regions of CD26 showed significant binding:

WWSPNGTFLAYAQ (SEQ ID NO: 148 corresponding to residues 215 to 227 ofSEQ ID NO: 144),

QLRCSGPGLPLYTLH (SEQ ID NO: 149 corresponding to residues 466 to 483 ofSEQ ID NO: 144)

LNETKFWYQMILP (SEQ ID NO: 150 corresponding to residues 519 to 531 ofSEQ ID NO: 144)

MGFVDNKRIAIWGWSY (SEQ ID NO: 151 corresponding to residues 616 to 631 ofSEQ ID NO: 144)

These 4 regions of CD26 appear to be spaced apart on the publishedcrystal structure and therefore may not all form a single discontinuousepitope for CDina26. Furthermore, based on the published CD26 crystalstructure, 3 of the 4 regions appear to be almost entirely buried insideCD26. The exception is WWSPNGTFLAYAQ (SEQ ID NO: 148), which is surfaceexposed at least at the PNGTF (SEQ ID NO: 152 corresponding to residues218 to 222 of SEQ ID NO: 144) residues.

Third, three distinct surface regions of CD26 were selected for analysisof discontinuous epitopes. Matrix 1 covers the catalytic area andN-terminal regions in proximity of the catalytic area (corresponding toresidues 260 to 400 of SEQ ID NO: 144). Matrix 2 covers the catalyticarea and C-terminal regions in proximity of the catalytic area thatpartially overlaps with set matrix1 (corresponding to residues 380 to538 of SEQ ID NO: 144). Finally, Matrix 3 covers a specific protrudingloop from CD26, which forms an immune-dominant structure (correspondingto residues 226 to 252 of SEQ ID NO: 144).

Compared to linear peptides, the discontinuous peptides showedrelatively lower signals, but the signals were more consistent. Whenresults obtained with all 3 matrices were taken together, multipleCDina26 binding regions of CD26 were identified.

Matrix 3, which focused on a protruding loop of CD26 corresponding toresidues 226 to 252 of SEQ ID NO: 144, did not identify any regions withsignificant CDina26 binding.

Matrix 1 (N-terminal of the focus region corresponding to residues 260to 400 of SEQ ID NO: 144) yielded 3 CDina26 binding regions:

DYDESSGRWNCLVAR (SEQ ID NO: 146 corresponding to residues 329 to 343 ofSEQ ID NO: 144).

DVTWATQERISLQWL (SEQ ID NO: 147 corresponding to residues 302 to 316 ofSEQ ID NO: 144)

TTGWVGRFRPSEPHF (SEQ ID NO: 153 corresponding to residues 350 to 364 ofSEQ ID NO: 144)

The strongest binding was observed for DYDESSGRWNCLVAR (SEQ ID NO: 146).When only peptides comprising DYDESSGRWNCLVAR (SEQ ID NO: 146) wereconsidered, the best binding peptides were those that also comprisedRFRPSEPHF (SEQ ID NO: 154 corresponding to residues 356 to 364 of SEQ IDNO: 144). RFRPSEPHF (SEQ ID NO: 154) includes the specific R358 residuementioned above. The specific additive effect on binding was consistentwith the CDina26 antibody targeting a discontinuous epitope thatincludes DYDESSGRWNCLVAR (SEQ ID NO: 146) and TTGWVGRFRPSEPHF (SEQ IDNO: 153). In another embodiment, the epitope further comprisesDVTWATQERISLQWL (SEQ ID NO: 147).

Matrix 2 yielded 4 binding regions:

TFITKGTWEVIG (SEQ ID NO: 155 corresponding to residues 395 to 406 of SEQID NO: 144)

DYLYYISNE (SEQ ID NO: 156 corresponding to residues 413 to 421 of SEQ IDNO: 144)

SCELNPERCQYY (SEQ ID NO: 157 corresponding to residues 446 to 457 of SEQID NO: 144)

SGPGLP (SEQ ID NO: 158 corresponding to residues 473 to 478 of SEQ IDNO: 144)

CDina26 binding to the Matrix 2 regions was weaker than binding toDYDESSGRWNCLVAR (SEQ ID NO: 146). Based on the published CD26 crystalstructure, regions DYLYYISNE (SEQ ID NO: 156) and SGPGLP (SEQ ID NO:158) appear to be mostly hidden inside the protein. Regions TFITKGTWEVIG(SEQ ID NO: 155) and SAELNPERCQYY (SEQ ID NO: 157) are surface exposedand accessible by an antibody.

A visual comparison of CDina26 binding to the 7 identified discontinuousbinding regions in CD26 the 7 is shown in FIG. 12 that displays theaverage binding to all discontinuous peptides that contain one of theidentified binding sequences. Here, DYDESSGRWNCLVAR (SEQ ID NO: 146) wasthe strongest binder, while DVTWATQERISLQWL (SEQ ID NO: 147) wasslightly stronger than TTGWVGRFRPSEPHF (SEQ ID NO: 153).

The strongest binding region, DYDESSGRWNCLVAR (SEQ ID NO: 146), ismostly exposed on the surface based on the published CD26 crystalstructure. Region TTGWVGRFRPSEPHF (SEQ ID NO: 153) is nearly completelyhidden inside the protein. Region DVTWATQERISLQWL (SEQ ID NO: 147) ispartly exposed on the surface at ATQER (SEQ ID NO: 159 corresponding toresidues 306 to 310 of SEQ ID NO: 144), and is located adjacent toDYDESSGRWNCLVAR (SEQ ID NO: 146).

The epitope mapping results were consistent with CDina26 specificallybinding a discontinuous epitope of CD26 comprising DYDESSGRWNCLVAR (SEQID NO: 146). In one embodiment, the discontinuous epitope furthercomprises one or both of DVTWATQERISLQWL (SEQ ID NO: 147) and PNGTF (SEQID NO: 152). While PNGTF (SEQ ID NO: 152) is conserved between human andpig CD26, the sequence differences in other components of the epitope(such as DYDESSGRWNCLVAR (SEQ ID NO: 146). which is not conservedbetween human and pig) is enough to abolish binding of CDina26 to thenative pig CD26. In another embodiment, the epitope further comprisesWWSPNGTFLAYAQ (SEQ ID NO: 148).

Example 9 Binding Affinity of CDina26 to CD26

Surface Plasmon Resonance system (Biacore®) was used to determine theaffinity of CDina26 for human CD26 according to standard protocols.Affinity measurements were taken at 25° C., immobilizing oncarboxymethyl dextran chip (CM5) 10.000 RU of an anti-IgG2b antibody (GEHealthcare. 22-0648-97 AC). CDina26 was prepared as stock in HBS-EP(0.01M HEPES, pH 7.4, 0.15M NaCl, 3 mM EDTA, 0.005% Surfactant P20)running buffer in a final concentration of 50 μg/ml, and for eachexperiment 2.000 RU were reversibly immobilized on the chip by injectionwith a flow of 10 μl/min in a period of 180 seconds.

Purified recombinant human CD26 (rhDipeptidyl peptidase IV; CreativeBioMart; CAT# DPP4-116H) was prepared in a stock concentration of5.8×10-6 M. CD26 was injected at increasing concentrations of 30×10⁻⁹,90×10⁻⁹ M, 270×10⁻⁹ M and 810×10⁻⁹ M. The CD26 samples were injectedwith a flow of 10 μl/minute; with HBS-EP buffer used as running buffer.A typical recording included a 3 minutes period of injection of the CD26followed by a period of 8 minutes of dissociation. The raw binding datawas analyzed according to standard methods. CM5 chip with immobilizedanti-IgG2b antibody was regenerated by injection of 10 mM Glycin-HCl pH1.7 with a flow of injection of 20 μl/min and for a period of 60seconds.

The results of the affinity measurements for CDina26 are shown below.Applicants believe that the CDina26 possesses superior bindingproperties compared to the prior art antibodies, and thus represent asignificant improvement over the prior art.

Ligand Ligand K_(on) K_(off) K_(D) CM5 Anti-IgG2b Analyte (1/Ms) (1/s)(M) Anti-IgG2b CDina26 Human 2.63E+04 1.32E−04 5.02e−09 CD26

Various modifications and variations of the described composition andmethod of the invention will be apparent to those in the art withoutdeparting from the scope and spirit of the invention. Although theinvention has been described in connection with specific embodiments, itshould be understood that the invention as claimed should not be undulylimited to such specific embodiments. Indeed, various modifications ofthe described modes for carrying out the invention which are obvious tothose in the relevant fields are intended to be within the scope of thefollowing claims.

REFERENCES

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The invention claimed is:
 1. An isolated monoclonal antibody thatspecifically binds human CD26 comprising a heavy chain variable regionand a light chain variable region, wherein a) the heavy chain variableregion comprises a VH CDR1 comprising the sequence of SEQ ID NO: 133, aVH CDR2 comprising the sequence of SEQ ID NO: 134, and a VH CDR3comprising the sequence of SEQ ID NO: 1; and b) the light chain variableregion comprises a VL CDR1 comprising the sequence of SEQ ID NO: 129 or131, a VL CDR2 comprising the sequence of SEQ ID NO: 130 or 132, and aVL CDR3 comprising the sequence of SEQ ID NO: 2 or
 3. 2. The antibody ofclaim 1, wherein the light chain variable region comprises an amino acidsequence having at least 90% sequence identity to SEQ ID NO: 4 or
 5. 3.The antibody of claim 1, wherein the heavy chain variable regioncomprises an amino acid sequence having at least 90% sequence identityto SEQ ID NO:
 26. 4. The antibody of claim 1, wherein the light chainvariable region comprises an amino acid sequence selected from the groupconsisting of SEQ ID NO: 4 to 21, and the heavy chain variable regioncomprises an amino acid sequence selected from the group consisting ofSEQ ID NO: 22 to
 47. 5. The antibody of claim 4, wherein the light chainvariable region comprises the amino acid sequence of SEQ ID NO: 4 or 5,and the heavy chain variable region comprises the amino acid sequence ofSEQ ID NO:
 26. 6. The antibody of claim 1, wherein the antibody has anantibody isotype selected from the group consisting of IgG1, IgG2, IgG3,IgG4, IgM, IgA, IgD and IgE.
 7. The antibody of claim 1, wherein theantibody has an antibody isotype of IgG 2B class.
 8. The antibody ofclaim 1, wherein the antibody is a recombinant antibody, a chimericantibody, a humanized antibody, a human antibody, an antibody fragment,a bispecific antibody, a monospecific antibody, or a monovalentantibody.
 9. The antibody of claim 1, wherein the antibody does notspecifically bind to porcine CD26.
 10. An antibody that that competesfor specific binding to human CD26 with an antibody comprising a lightchain variable region of SEQ ID NO: 4 or 5, and a heavy chain variableregion of SEQ ID NO:
 26. 11. A pharmaceutical composition comprising theantibody of claim 1 and a pharmaceutically acceptable excipient.
 12. Amethod of preventing or treating Graft-versus-Host disease (GvHD) in asubject in need thereof, comprising administering a therapeuticallyeffective dose of the antibody of claim
 1. 13. A kit comprising theantibody of claim
 1. 14. A hybridoma cell line deposited at CBA-ICLC ofGenoa (Italy) as PD 12002 or a subclone of the hybridoma, wherein thehybridoma or subclone thereof produces an antibody that specificallybinds human CD26.
 15. An antibody produced by the hybridoma cell line orsubclone of claim 14, wherein the antibody specifically binds humanCD26.
 16. An isolated composition comprising an antibody mixture,wherein the antibody mixture comprises or consists of the antibodiesproduced by the hybridoma cell line or subclone of claim 14, and whereinthe antibody mixture specifically binds human CD26.
 17. An isolatedantibody that specifically binds human CD26 comprising a heavy chainvariable region and a light chain variable region, wherein the heavy andlight chain variable regions comprise the three VH CDR sequences andthree VL CDR sequences, respectively, of an antibody produced by thehybridoma cell line or subclone of claim
 14. 18. The kit of claim 13,further comprising a) at least one immunosuppressive drug, or b) atleast one corticosteroid and at least one antihistamine.
 19. Theantibody of claim 1, wherein the light chain variable region comprises aVL CDR1 comprising the sequence of SEQ ID NO: 129, a VL CDR2 comprisingthe sequence of SEQ ID NO: 130, and a VL CDR3 comprising the sequence ofSEQ ID NO:
 2. 20. The antibody of claim 19, wherein the antibody has anantibody isotype selected from the group consisting of IgG1, IgG2, IgG3,IgG4, IgM, IgA, IgD and IgE.
 21. The antibody of claim 19, wherein theantibody has an antibody isotype of IgG 2B class.
 22. The antibody ofclaim 19, wherein the antibody is a recombinant antibody, a chimericantibody, a humanized antibody, an antibody fragment, a bispecificantibody, a monospecific antibody, or a monovalent antibody.
 23. Theantibody of claim 19, wherein the antibody is a humanized antibody. 24.A pharmaceutical composition comprising the antibody of claim 19 and apharmaceutically acceptable excipient.
 25. A method of preventing ortreating GvHD in a subject in need thereof, comprising administering atherapeutically effective dose of the antibody of claim
 19. 26. Theantibody of claim 5, wherein the light chain variable region comprisesthe amino acid sequence of SEQ ID NO:
 4. 27. The antibody of claim 26,wherein the antibody is a chimeric antibody.
 28. A pharmaceuticalcomposition comprising the antibody of claim 26 and a pharmaceuticallyacceptable excipient.
 29. A method of preventing or treating GvHD in asubject in need thereof, comprising administering a therapeuticallyeffective dose of the antibody of claim 26.